Live bacterial vaccines for viral infection prophylaxis or treatment

ABSTRACT

A live bacterium, having a DNA construct stabilized against transduction of other bacteria, having a promoter sequence and encoding a fusion peptide, comprising a bacterial secretion peptide portion and a non-bacterial immunogenic polypeptide portion, having a nucleotide sequence coding for the non-bacterial immunogenic polypeptide portion which has at least one codon optimized for bacterial expression. The bacterium has a secretion mechanism which interacts with at least the bacterial secretion peptide portion to cause a secretion of the fusion peptide from the bacterium, and a genetic virulence attenuating mutation. The bacterium is adapted to act as an animal vaccine, to transiently infect a tissue of the animal, and cause an immunity response to the non-bacterial immunogenic polypeptide portion in the animal to a non-bacterial organism associated with the non-bacterial immunogenic polypeptide portion.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/859,569, filed Sep. 21, 2007, which claims benefit ofpriority from Provisional Patent Application 60/826,542, filed Sep. 22,2006.

FIELD OF THE INVENTION

This invention is generally in the field of live bacterial vaccines forviral infection prophylaxis or treatment.

BACKGROUND OF THE INVENTION

Citation or identification of any reference herein, or any section ofthis application shall not be construed as an admission that suchreference is available as prior art to the present application.

There are three types of influenza viruses Influenza A, B, and C.Influenza types A or B viruses cause epidemics of disease almost everywinter. In the United States, these winter influenza epidemics can causeillness in 10% to 20% of people and are associated with an average of36,000 deaths and 114,000 hospitalizations per year. Influenza type Cinfections cause a mild respiratory illness and are not thought to causeepidemics. Influenza type A viruses are divided into subtypes based ontwo proteins on the surface of the virus. These proteins are termedhemagglutinin (H) and neuraminidase (N). Influenza A viruses are dividedinto subtypes based on these two proteins. There are 16 differenthemagglutinin subtypes H1, H2, H3, H4, H6, H7, H8, H9 H10 H11 H12, H13,H14, H15 or H16 and 9 different neuraminidase subtypes N1 N2 N3 N4 N5 N6N7 N8 or N9, all of which have been found among influenza A viruses inwild birds. Wild birds are the primary natural reservoir for allsubtypes of influenza A viruses and are thought to be the source ofinfluenza A viruses in all other animals. The current subtypes ofinfluenza A viruses found in people are A(H1N1) and A(H3N2). Influenza Bvirus is not divided into subtypes.

In 1918, a new highly pathogenic influenza H1N1 pandemic swept theworld, killing an estimated 20 and 50 million people. The H1N1 subtypecirculated from 1918 until 1957 which then was replaced by viruses ofthe H2N2 subtype, which continued to circulate until 1968. Since 1968,H3N2 viruses have been found in the population. Because H1N1 virusesreturned in 1977, two influenza A viruses are presently cocirculating(Palese and Garcia-Sarstre J Clin Invest, July 2002, Volume 110, Number1, 9-13). The pathogenicity of the initial 1918 H1N1 has not beenequaled by any of the latter H1N1, H2N2 or H3N2 subtypes, althoughinfection from some subtypes can be severe and result in death. Bymolecular reconstruction, the genome of the 1918 flu including the aminoacid sequences of the H1 and N1 antigens is now known (Kaiser, Science310: 28-29, 2005; Tumpey et al., Science 310: 77-81, 2005).

In 1997, 2003, and again in 2004, antigenically-distinct avian H5N1influenza viruses emerged as pandemic threats to human beings. Duringeach of these outbreaks there was concern that the avian viruses wouldadapt to become transmissible from human to human. Furthermore,oseltamivir (Tamiflu®) was ineffective in 50% of avian influenzapatients in Thailand (Tran et al. N. Engl. J. Med 350: 1179, 2004) and anew mutation in the neuraminidase has been identified which causesresistance to oseltamivir. Sequence analysis of the neuraminidase generevealed the substitution of tyrosine for histidine at amino acidposition 274 (H274Y), associated with high-level resistance tooseltamivir in influenza (N1) viruses (Gubareva et al., Selection ofinfluenza virus mutants in experimentally infected volunteers treatedwith oseltamivir. J Infect Dis 2001; 183:523-531; de Jong et al.,Oseltamivir Resistance during Treatment of Influenza A (H5N1) Infection.N. Engl. J. Med. 353:2667-2672, 2005). Such changes may alter theantigenic nature of the protein and reduce the effectiveness of vaccinesnot matched to the new variant. Other avian influenza strains ofpotential danger include H1N1, H7N7 and H9N2.

The optimum way of dealing with a human pandemic virus would be toprovide a clinically approved well-matched vaccine (i.e., containing thehemagglutinin and/or neuraminidase antigens of the emerging humanpandemic strain), but this cannot easily be achieved on an adequatetimescale because of the time consuming method of conventional influenzavaccine production in chicken eggs.

2.1 Live Bacterial Vaccine Vectors

Live attenuated bacterial vaccine vectors offer an important alternativeto conventional chicken egg based vaccines. Growth on embryonated heneggs, followed by purification of viruses from allantoic fluid, is themethod by which influenza virus has traditionally been grown for vaccineproduction. More recently, viruses have been grown on cultured celllines, which avoids the need to prepare virus strains that are adaptedto growth on eggs and avoids contamination of the final vaccine with eggproteins. However, because some of the vaccine virus may be produced incanine tumor cells (e.g., MDCK), there is concern for contamination ofthe vaccine by cancer causing elements. Moreover, both must undergo alabor intensive and technically challenging purification process, with atotal production time of 3 to 6 months. Because of the time factors andscale-up, these vaccines are produced in large, but finite batches.Meeting a world-wide demand requires stockpiling of multiple batches.Therefore, traditionally produced vaccine produced before a pandemic,would likely be generated based upon an avian influenza virus and itsantigens more than a year earlier and therefore may not be well matchedto an emerging variant and could result in only partial protection.Bacterial vectors self replicate in simple growth media can be producedextremely rapidly by virtue of exponential growth and require minimalpurification such as a single centrifugation and resuspension in apharmaceutically acceptable excipient.

Human studies have shown that antibody titres against hemagglutinin ofhuman influenza virus are correlated with protection (a serum samplehemagglutination-inhibition titre of about 30-40 gives around 50%protection from infection by a homologous virus) (Potter & Oxford (1979)Br Med Bull 35: 69-75). Antibody responses are typically measured byenzyme linked immunosorbent assay (ELISA), immunoblotting,hemagglutination inhibition, by microneutralisation, by single radialimmunodiffusion (SRID), and/or by single radial hemolysis (SRH). Theseassay techniques are well known in the art.

Salmonella bacteria have been recognized as being particularly useful aslive “host” vectors for orally administered vaccines because thesebacteria are enteric organisms that, when ingested, can infect andpersist in the gut (especially the intestines) of humans and animals.

As a variety of Salmonella bacteria are known to be highly virulent tomost hosts, e.g., causing typhoid fever or severe diarrhea in humans andother mammals, the virulence of Salmonella bacterial strains toward anindividual that is targeted to receive a vaccine composition must beattenuated. Attenuation of virulence of a bacterium is not restricted tothe elimination or inhibition of any particular mechanism and may beobtained by mutation of one or more genes in the Salmonella genome(which may include chromosomal and non-chromosomal genetic material).Thus, an “attenuating mutation” may comprise a single site mutation ormultiple mutations that may together provide a phenotype of attenuatedvirulence toward a particular host individual who is to receive a livevaccine composition for Avian Influenza. In recent years, a variety ofbacteria and, particularly, serovars of Salmonella enterica, have beendeveloped that are attenuated for pathogenic virulence in an individual(e.g., humans or other mammals), and thus proposed as useful fordeveloping various live bacterial vaccines (see, e.g., U.S. Pat. Nos.5,389,368; 5,468,485; 5,387,744; 5,424,065; Zhang-Barber et al.,Vaccine, 17; 2538-2545 (1999); all expressly incorporated herein byreference). In the case of strains of Salmonella, mutations at a numberof genetic loci have been shown to attenuate virulence including, butnot limited to, the genetic loci phoP, phoQ, cdt, cya, crp, poxA, rpoS,htrA, nuoG, pmi, pabA, pts, damA, purA, purB, purI, zwf, aroA, aroC,gua, cadA, rfc, rjb, rfa, ompR, msbB and combinations thereof.

Bacterial flagella are known to be antigenic and subject to antigenic orphase variation which is believed to help a small portion of thebacteria in escaping the host immune response. The bacterial flagellarantigens are referred to as the H1 and H2 antigens. To avoid confusionwith the viral hemagglutinin H antigen, the bacterial flagellar Hantigen will be referred to as fH henceforth. Because theSalmonella-based vaccination of a heterologous antigen is dependent uponthe bacteria's ability to colonize the gut, which may be reduced do tothe initial immune response, the vaccination ability of the secondimmunization may be diminished due to an immune response to the vector.In Salmonella, Hin invertase belongs to the recombinase family, whichincludes Gin invertase from phage Mu, Cin invertase from phage P1, andresolvases from Tn3 and the transposon (Glasgow et al. 1989, p. 637-659.In, D. E. Berg, and M. M. Howe (ed.), Mobile DNA. American Society forMicrobiology, Washington, D.C.). Hin promotes the inversion of achromosomal DNA segment of 996 bp that is flanked by the 26-bp DNAsequences of hixL and hixR (Johnson and Simon. 1985. Cell 41:781-791).Hin-mediated DNA inversion in S. typhimurium leads to the alternativeexpression of the fH1 and fH2 flagellin genes known as phase variation.Hin (21 kDa) exists in solution as a homodimer and binds to hix sites asa dimer (Glasgow et al. 1989. J. Biol. Chem. 264:10072-10082). Inaddition to Hin and the two hix sites, a cis-acting DNA sequence(recombinational enhancer) and its binding protein (F is, 11 kDa) arerequired for efficient inversion in vitro (Johnson et al. 1986. Cell46:531-539). Live Salmonella vaccines have not had deletions of the hingene nor defined fH1 or fH2 antigens, nor have they been constructedsuch that they lack fH antigens altogether. Accordingly, live Salmonellavaccines have not been constructed to maximize a prime-boost strategywhich alternates or eliminates the fH antigen whereby the immuneresponse of the fH antigen of the first immunization (prime) is notspecific for the antigen of the second immunization (boost). Therefore,the boost immunization is not diminished by a rapid elimination by theimmune system, and is therefore able to persist longer and moreeffectively present the immunizing antigen.

Introduction of viral genes into bacteria results in geneticallyengineered microorganisms (GEMs) for which there may be concernregarding containment of the introduced gene in the environment and itsability to reassort. Such genes could in theory provide virulencefactors to non-pathogenic or less pathogenic viral strains if allowed torecombine under circumstances were the bacterial vaccine could co-occurat the same time in the same individual as a wild type viral infection.Thus, methods that reduce bacterial recombination and increase bacterialgenetic isolation are desirable.

Insertion sequences (IS) are genetic elements that can insert copies ofthemselves into different sites in a genome. These elements can alsomediate various chromosomal rearrangements, including inversions,deletions and fusion of circular DNA segments and alter the expressionof adjacent genes. IS200 elements are found in most Salmonella species.S. typhimurium strain LT2 has six IS200s. Salmonella typhimurium strain14028 has been described to possess an additional IS200 element atcentisome 17.7 which is absent in other commonly studied Salmonellastrains LT2 and SL1344 (Murray et al., 2004 Journal of Bacteriology,186: 8516-8523). These authors describe a spontaneous hot spot (highfrequency) deletion of the Cs 17.7 to Cs 19.9 region. Live Salmonellavaccines have not had deletions of IS200 elements which would limit suchrecombination events.

Salmonella strains are known to possess phage and prophage elements.Such phage are often capable of excision and infection of othersusceptible strains and are further capable of transferring genes fromone strain by a process known as transduction. Live Salmonella vaccineshave not had deletions in phage elements such as phage recombinaseswhich exist in Salmonella, such that the phage are no longer capable ofexcision and reinfection of other susceptible strains.

Salmonella strains are known to be capable of being infected by bacteriaphage. Such phage have the potential to carry genetic elements from oneSalmonella strain to another. Live Salmonella vaccines have notcomprised mechanisms to limit phage infection such as the implantationand constitutive expression of the P22 phage repressor C2.

Bacterial expression of the viral hemagglutinin genes was firstdescribed by Heiland and Gething (Nature 292: 581-582, 1981) and Daviset al., (Proc. Natl. Acad. Sci. USA 78: 5376-5380). These authorssuggest that the recombinant protein could be used as a vaccine withoutregard to the fact that the viral genetic loci are not optimal forbacterial expression. These authors did not suggest the use of livebacterial vectors as vaccine carriers, such as the geneticallystabilized and isolated vectors of the present application, nor the useof defined flagellar antigens or no flagellar antigens. Nor did theseauthors suggest the use of secreted proteins.

Use of secreted proteins in live bacterial vectors has been demonstratedby several authors. Holland et al. (U.S. Pat. No. 5,143,830, expresslyincorporated herein by reference) have illustrated the use of fusionswith the C-terminal portion of the hemolysin A (hlyA) gene. Whenco-expressed in the presence of the hemolysin protein secretion channel(hlyBD), heterologous fusions are readily secreted from the bacteria.Similarly, Galen et al. (Infection and Immunity 2004 72: 7096-7106) haveshown that heterologous fusions to the ClyA are secreted andimmunogenic. Other heterologous protein secretion systems include theuse of the autotransporter family. For example, Veiga et al. (2003Journal of Bacterilogy 185: 5585-5590) demonstrated hybrid proteinscontaining the b-autotransporter domain of the immunoglobulin A (IgA)protease of Nisseria gonorrhoea.

Bacterial expression of the viral hemagglutinin genes was firstdescribed by Heiland and Gething (Nature 292: 581-582, 1981) and Daviset al., (Proc. Natl. Acad. Sci. USA 78: 5376-5380). These authors teachthat the antigens may be purified from the bacteria in order to be usedas vaccines and did not suggest the use of live attenuated bacterialvectors. Furthermore, the codon usage of the viral genome is not optimalfor bacterial expression. Accordingly, a gram-negative bacterium of theenterobacteraceae such as E. coli and Salmonella will have a differentcodon usage preference (National Library of Medicine, National Centerfor Biotechnology Information, GenBank Release 150.0 [Nov. 25, 2005])and would not be codon optimized. Further, these authors usedantibiotic-containing plasmids and did not use stable chromosomallocalization. Nor did these authors suggest heterologous fusions inorder for the bacteria to secrete the antigens.

Kahn et al. (EP No. 0863211) have suggested use of a live bacterialvaccine with in vivo induction using the E. coli nitrite reductasepromoter nirB. These authors further suggest that the antigenicdeterminant may be an antigenic sequence derived from a virus, includingInfluenza virus. However, Khan et al. did not describe a vaccine foravian influenza virus. They did not describe the appropriate antigensfor an Avian Influenza virus, the hemaglutinin and neuraminidase, anddid not describe how to genetically match an emerging Avian Influenzavirus. Furthermore, it has become apparent that certain assumptions, andexperimental designs described by Khan et al. regarding live AvianInfluenza vaccines would not be genetically isolated or have improvedgenetic stability in order to provide a live vaccine for Avian Influenzathat would be acceptable for use in humans. For example, Khan et al.state that any of a variety of known strains of bacteria that have anattenuated virulence may be genetically engineered and employed as livebacterial carriers (bacterial vectors) that express antigen polypeptidesto elicit an immune response including attenuated strains of S.typhimurium and, for use in humans, attenuated strains of S. typhi(i.e., S. enterica serovar Typhi). In support of such broad teaching,they point to the importance of “non-reverting” mutations, especiallydeletion mutations which provide the attenuation. However, non-reversiononly refers to the particular gene mutated, and not to the genome per sewith its variety of IS200, phage and prophage elements capable of avariety of genetic recombinations and/or even transductions to otherbacterial strains. Khan et al. did not describe a bacterial strain withimproved genetic stability, nor methods to reduce genetic recombination,such as deletion of the IS200 elements. Khan et al. did not describe abacterial strain with improved genetic stability by deletion of thebacteria phage and prophage elements nor limiting their transducingcapacity. Neither did Khan et al. describe methods to minimize bacterialgenetic exchange, such as constitutive expression of the P22 C2 phagerepressor.

The above comments illustrate that Khan et al. have not provided thefield with an effective vaccine against avian influenza. Clearly, needsremain for an genetically isolated and genetically stable, orallyadministered vaccine against Avian Influenza which is capable of rapidgenetically matching an emerging pathogenic variant.

SUMMARY OF THE INVENTION

The present invention provides live attenuated bacterial strains thatexpress one or more immunogenic polypeptide antigens of a virus,preferably an Avian Influenza virus, that is effective in raising animmune response in mammals.

In particular, one aspect of the invention relates to live attenuatedbacterial strains which may include Salmonella vectoring avian influenzaantigens that can be administered orally to an individual to elicit animmune response to protect the individual from Avian Influenza.

The preferred bacteria are serovars of Salmonella. Preferably, thebacteria are genetically isolated from infecting bacteria phage and haveimproved genetic stability by virtue of deletion of IS200 and phageelements. The preferred Salmonella strains of the invention areattenuated by mutations at genetic loci which, alone or in combination,provides sufficient attenuation, and defined flagellar antigens for animproved prime/boost strategy. The attenuating mutations may be those ofstrains known to exhibit a degree of safety in humans including but notlimited to Ty21a, CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800,holavax, M01ZH09 or VNP20009 or may be novel combinations of mutations.

Whereas the current medical practice uses derivatives of pathogenicavian strains in chicken eggs to provide vaccines that generate animmune response including antibodies in humans or other mammals againstknown pathogenic avian strains, the invention provides methods andcompositions comprising genetically isolated bacterial vectors withenhanced genetic stability vectoring Avian Influenza virus antigens toprotect against emerging pathogenic human strains.

Furthermore, whereas the prior art often fails to achieving closeantigenic match between the vaccine strain and the target strain, theinvention targets viruses for vaccine strains based on their emergingpathogenicity, and produces an effective vaccine more closely matched tothe antigen profile of the emerging pathogen. As the invention requiresdetailed knowledge of the antigenic profile of an emerging strain, sucha vaccine can be produced at the time of need in order to reduce therisk of an unmatched vaccine and potential effects of partial protectionin a human pandemic outbreak. Thus the invention provides vaccines forprotecting a human patient against infection by an emerging AvianInfluenza virus strain.

Preferably, the vaccines according to the present invention comprisegenetically stable bacterial vectors carrying one or more antigen froman Avian Influenza virus strain that can cause highly pathogenic AvianInfluenza.

The invention further preferably provides for vaccines againstoseltamivir resistant strains.

Accordingly, when orally administered to an individual, a liveSalmonella bacterial vaccine, in accordance with the present invention,that is genetically engineered to express one or more Avian Influenzaantigens as described herein have the inherent ability to establish apopulation (infection) in the gut and, if properly modified they couldprovide a desirable source of immunogenic Avian Influenza antigenpolypeptide(s) to elicit an immune response in the mucosal tissue of theindividual.

The antigen(s) can invoke an antibody response in the patient that iscapable of neutralizing the emerging avian influenza vaccine strainswith high efficiency, as well as emerging heterologous Avian Influenzavaccine strains, with moderate efficiency. Preferably, the emergingavian influenza vaccine will be within the same hemagglutinin and orneuraminidase type (i.e., H1, H5, H5 (H274Y), H7 or H9 and/or N1, N2 orN7) as are the current pathogenic avian influenza strains.

The live vaccine compositions are suitable for oral administration to anindividual to provide protection from avian influenza. Preferably, avaccine composition comprises a suspension of a live bacterial straindescribed herein in a physiologically accepted buffer or saline solutionthat can be swallowed from the mouth of an individual. However, oraladministration of a vaccine composition to an individual may alsoinclude, without limitation, administering a suspension of a bacterialvaccine strain described herein through a nasojejunal or gastrostomytube and administration of a suppository that releases a live bacterialvaccine strain to the lower intestinal tract of an individual. Vaccinesof the invention may be formulated for delivery by other various routese.g. by intramuscular injection, subcutaneous delivery, by intranasaldelivery (e.g. WO00/47222, U.S. Pat. No. 6,635,246), intradermaldelivery (e.g. WO02/074336, WO02/067983, WO02/087494, WO02/0832149WO04/016281) by transdermal delivery, by transcutaneous delivery, bytopical routes, etc. Injection may involve a needle (including amicroneedle), or may be needle-free.

Annual human Influenza vaccines typically include more than oneInfluenza strain, with trivalent vaccines being normal (e.g. twoInfluenza A virus antigens, and one Influenza B virus antigen). Inpandemic years, however, a single monovalent strain may be used. Thusthe pathogenic avian antigen(s) described above may be the soleInfluenza antigen(s) in a vaccine of the invention, or the vaccine mayadditionally comprise antigen(s) from one or more (e.g. 1, 2, 3, 4 ormore) annual Influenza virus strains. Specific vaccines of the inventionthus include: (i) a vaccine comprising the pathogenic avian antigen(s)as the sole Influenza antigen(s); (ii) a vaccine comprising thepathogenic avian antigen(s) plus antigen(s) from another pathogenicAvian Influenza strain (e.g., H1N1, H5N1, H7N7, H2N9, H9N2).

Vaccines of the invention use one or more avian antigens to protectpatients against infection by an Influenza virus strain that is capableof human-to-human transmission i.e. a strain that will spreadgeometrically or exponentially within a given human population withoutnecessarily requiring physical contact. The patient may also beprotected against strains that infect and cause disease in humans, butthat are caught from birds rather than from other humans (i.e., bird tohuman transmission). The invention is particularly useful for protectingagainst infection by pandemic, emerging pandemic and future panderinghuman strains e.g. for protecting against H5 and N1 influenza subtypes.Depending on the particular season and on the nature of the antigenincluded in the vaccine, however, the invention may protect against anyhemagglutinin subtypes, including H1, H2, H3, H4, H5, H6, H7, H8, H9,H10, H11, H12, H13, H14, H15 or H16 or various neuraminidase subtypes,including N1, N2, N3, N4, N5, N6, N7, N8 or N9.

The characteristics of an Influenza strain that give it the potential tocause a pandemic outbreak may include: (a) it contains a new orantigenically altered hemagglutinin compared to the hemagglutinins incurrently-circulating human strains i.e., one that has not been evidentin the human population for over a decade (e.g. H2), or has notpreviously been seen at all in the human population (e.g. H5, H6 or H9,that have generally been found only in bird populations), such that thehuman population will be immunologically naive to the strain'shemagglutinin or that is a subtype which is antigenically altered bychanges in amino acid sequence or glycosylation; (b) it is capable ofbeing transmitted horizontally in the human population; (c) is capableof being transmitted from animals (including birds, dogs, pigs) tohumans; and/or (d) it is pathogenic to humans.

As a preferred embodiment of the invention protects against a strainthat is capable of human-to-human or bird-to-human or bird-to-birdtransmission, one embodiment of the invention in accordance with thataspect will generally include at least one gene that originated in amammalian (e.g. in a human) Influenza virus and one gene whichoriginated in a bird or non-human vertebrate. Vaccines in accordancewith various aspects of the invention may therefore include an antigenfrom an Avian Influenza virus strain. This strain is typically one thatis capable of causing highly pathogenic Avian Influenza (HPAI). HPAI isa well-defined condition (Alexander Avian Dis (2003) 47(3 Suppl):976-81)that is characterized by sudden onset, severe illness and rapid death ofaffected birds/flocks, with a mortality rate that can approach 100%. Lowpathogenicity (LPAI) and high pathogenicity strains are easilydistinguished e.g. van der Goot et al. (Epidemiol Infect (2003)131(2):1003-13) presented a comparative study of the transmissioncharacteristics of low and high pathogenicity H5N2 avian strains. Forthe 2004 season, examples of HPAI strains are H5N1 Influenza A virusese.g. A/Viet Nam/I 196/04 strain (also known as A Vietnam/3028/2004 orA/Vietnam/3028/04). The skilled person will thus be able to identify orpredict future HPAI strains and the DNA sequence and amino acidcompositions of the H and N antigens as and when they emerge. The AvianInfluenza strain may be of any suitable hemagglutinin subtype, includingH1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.The Avian Influenza strain may further be of any suitable neuraminidasesubtype N1, N2, N3, N4, N5, N6, N7, N8, or N9. The vaccines of theinvention may comprise two or more (i.e., two, three, four, or five)Avian Influenza hemagglutinin and neuraminidase antigens. Such AvianInfluenza strains may comprise the same or different hemagglutininsubtypes and the same or different neuraminidase subtypes.

A preferred vaccine composition will contain a sufficient amount of livebacteria expressing the antigen(s) to produce an immunological responsein the patient. Accordingly, the attenuated Salmonella strains describedherein are both safe and useful as live bacterial vaccines that can beorally administered to an individual to provide immunity to AvianInfluenza and, thereby, protection from Avian Influenza.

Although not wishing to be bound by any particular mechanism, aneffective mucosal immune response to Avian Influenza antigen(s) inhumans by oral administration of genetically engineered, attenuatedstrains of Salmonella strains as described herein may be due to theability of such mutant strains to persist in the intestinal tract. Eachbacterial strain useful in the invention carries an antigen-expressingplasmid or chromosomally integrated cassette that encodes and directsexpression of one or more Avian Influenza antigens of Avian Influenzavirus when resident in an attenuated Salmonella strain described herein.As noted above, Avian Influenza antigens that are particularly useful inthe invention include an H1, H5, H5 (H274Y), H7 or H9 antigenpolypeptide (or immunogenic portion thereof), a N1, N2 or N7 antigenpolypeptide (or immunogenic portion thereof), and a fusion polypeptidecomprising a heterologous secretion peptide linked in-frame to theantigenic peptide.

The serovars of S. enterica that may be used as the attenuated bacteriumof the live vaccine compositions described herein include, withoutlimitation, Salmonella enterica serovar Typhimurium (S. typhimurium),Salmonella montevideo, Salmonella enterica serovar Typhi (S. typhi),Salmonella enterica serovar Paratyphi B (S. paratyphi 13), Salmonellaenterica serovar Paratyphi C (S. paratyphi C), Salmonella entericaserovar Hadar (S. hadar), Salmonella enterica serovar Enteriditis (S.enteriditis), Salmonella enterica serovar Kentucky (S. kentucky),Salmonella enterica serovar Infantis (S. infantis), Salmonella entericaserovar Pullorurn (S. pullorum), Salmonella enterica serovar Gallinarum(S. gallinarum), Salmonella enterica serovar Muenchen (S. muenchen),Salmonella enterica serovar Anaturn (S. anatum), Salmonella entericaserovar Dublin (S. dublin), Salmonella enterica serovar Derby (S.derby), Salmonella enterica serovar Choleraesuis var. kunzendorf (S.cholerae kunzendorf), and Salmonella enterica serovar minnesota (S.minnesota).

By way of example, live Avian Influenza vaccines in accordance withaspects of the invention include known strains of S. enterica serovarTyphimurium (S. typhimurium) and S. enterica serovar Typhi (S. typhi)which are further modified as provided by the invention to form suitablevaccines for the prevention and treatment of Avian Influenza. SuchStrains include Ty21a, CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800,aroA-/serC-, holavax, M01ZH09, VNP20009.

Novel strains are also encompassed that are attenuated in virulence bymutations in a variety of metabolic and structural genes. The inventiontherefore may provide a live vaccine composition for protecting againstAvian Influenza comprising a live attenuated bacterium that is a serovarof Salmonella enterica comprising, an attenuating mutation in a geneticlocus of the chromosome of said bacterium that attenuates virulence ofsaid bacterium and wherein said attenuating mutation is the Suwwandeletion (Murray et al., 2004) or combinations with other knownattenuating mutations. Other attenuating mutation useful in theSalmonella bacterial strains described herein may be in a genetic locusselected from the group consisting of phoP, phoQ, edt, cya, crp, poxA,rpoS, htrA, nuoG, pmi, pabA, pts, damA, purA, purB, purI, zwf, purF,aroA, aroB, aroC, aroD, serC, gua, cadA, rfc, rjb, rfa, ompR, msbB andcombinations thereof.

The invention may also provide a process for preparing geneticallystable bacterial vaccines for protecting a human patient againstinfection by an Avian Influenza virus strain, comprising geneticallyengineering the avian antigen from an Avian Influenza virus strain thatcan cause highly pathogenic Avian Influenza to comprise a bacteriallycodon optimized expression sequence within a bacterial plasmidexpression vector or chromosomal localization expression vector andfurther containing engineered restriction endonuclease sites such thatthe bacterially codon optimized expression gene contains subcomponentswhich are easily and rapidly exchangeable in order to facilitate rapidexchange of the genetic subcomponents to achieve a well matched antigento the emerging Avian Influenza pathogen. The plasmid and/or chromosomalexpression constructs may be further modified to result in the secretionof the viral antigens. Administration of the vaccine to the patientinvokes an antibody response that is capable of neutralizing said AvianInfluenza virus strain.

The invention may also provide methods and compositions for producing abacterial vector expressing one or more Avian Influenza antigens wheresaid bacterial vector has one or more deletions in IS200 elements whichresults in enhance genetic stability. The composition and methodscomprise a bacterial strain with a deletion in the IS200 elements, suchthat the bacteria are no longer capable of genetic rearrangement usingIS200 elements. Such a deletion is generated in any one or more IS200element, which is then confirmed using standard genetic techniques.

The invention may also provide methods and compositions for producing agenetically stabilized bacterial vector expressing one or more AvianInfluenza antigens where said bacterial vector has one or more deletionsin bacteria phage or prophage elements which enhanced genetic stabilityand prevent phage excision. The composition and methods comprise abacterial strain with one or more deletions in bacteria phage orprophage elements, such that the bacteria are no longer capable ofgenetic rearrangement using bacteria phage or prophage elements. Such adeletion is generated in any bacteria phage or prophage elements, whichis then confirmed using standard genetic techniques. Such strains havephage with reduced capacity for transduction of genes to other strains.

The invention may also provide methods and compositions for producing abacterial vector expressing one or more avian influenza antigens wheresaid bacterial vector constitutively expresses the P22 phage C2repressor, thereby preventing new infections by bacteria phage andfurther preventing subsequent phage transductions by these phage.

Live Salmonella vaccines have not had deletions of the hin gene nordefined fH1 or fH2 antigens, nor have they been constructed such thatthey lack fH antigens altogether. Accordingly, prior live Salmonellavaccines have not been constructed to maximize a prime-boost strategywhich alternates or eliminates the fH antigen whereby the immuneresponse of the fH antigen of the first immunization (prime) is notspecific for the antigen of the second immunization (boost). Therefore,the boost immunization is not diminished by a rapid elimination by theimmune system, and is therefore able to persist longer and moreeffectively present the immunizing heterologous Avian Influenza antigen.

An embodiment of the present invention therefore may also providemethods and compositions for producing a bacterial vector expressing oneor more Avian Influenza antigens where said bacterial vector has adefined flagellar H antigen (1H). The composition and methods comprise abacterial strain with a deletion in the Hin recombinase gene, such thatthe bacteria are no longer capable of alternating between fH1 and fH2antigens. Such a deletion is generated in either an fH1 or fH2serologically defined strain, which is then reconfirmed followingdeletion or disruption of the hin recombinase gene. The inventionfurther provides methods and compositions for producing a bacterialvector which lacks flagellar antigens generated by deletion of the fliBCgenes (i.e., fH0). Therefore, an improved composition for a prime/booststrategy is provided where the second vaccination comprisesadministration of a vaccine where the fH antigen composition isdifferent from the first vaccination.

The invention may also provide a method for protecting a human patientagainst infection by an Avian Influenza virus strain with an improvedprime/boost strategy, comprising the step of administering to thepatient a vaccine that comprises an antigen from an Avian Influenzavirus strain that can cause highly pathogenic Avian Influenza or 1918influenza within a bacterial vector expressing one or more AvianInfluenza antigens where said bacterial vector has a defined fH antigenor no fH antigen (i.e., fH1, fH2, or fH0). The invention may furtherprovide a method of administering a second bacterial vector expressingone or more Avian Influenza antigens comprising a second step where thesecond administration where said bacterial vector has a defined fHantigen which is different fH antigen composition than the fH antigen ofthe first administration or no fH antigen. The second administrationincludes a bacterial vaccine where the first vaccine administration is abacterial vaccine of the present invention or is another vaccine notencompassed by the present application, e.g., another bacterial vaccineor an egg-based vaccine.

Similarly, the invention may also provide a kit comprising (a) a firstcontainer comprising a bacterial expression codon optimized antigen froma pathogenic Avian Influenza virus strain containing unique geneticallyengineered restriction sites contained within either a bacterial proteinexpression plasmid or a bacterial chromosomal protein expression vectorand (b) a second container comprising bacterial vector(s) with one ormore (e.g., fH1, fH2 or fH0) flagellar antigen(s). Component (a) will bemodifiable to genetically match an emerging Avian Influenza virus usingstandard in vitro molecular techniques and can be combined withcomponent (b) to generate one or more bacterial strains with definedflagellar antigens which constitute a live vaccine. The variation(s) inflagellar antigens provided by the kit provide for more than one livevaccine strain in which a first immunization (prime) using one strainmay be followed at an appropriate time such as 2 to 4 weeks by a secondimmunization (boost) using a second strain with a different fH antigenor no fH antigen. The live vaccine compositions are suitable for oraladministration to an individual to provide protection from AvianInfluenza.

Preferably, a vaccine composition comprises a suspension of a livebacterial strain described herein in a physiologically accepted bufferor saline solution that can be swallowed from the mouth of anindividual. However, oral administration of a vaccine composition to anindividual may also include, without limitation, administering asuspension of a bacterial vaccine strain described herein through anasojejunal or gastrostomy tube and administration of a suppository thatreleases a live bacterial vaccine strain to the lower intestinal tractof an individual.

4. Definitions

In order that the invention may be more fully understood, the followingterms are defined.

As used herein, “attenuated”, “attenuation”, and similar terms refer toelimination or reduction of the natural virulence of a bacterium in aparticular host organism, such as a mammal. “Virulence” is the degree orability of a pathogenic microorganism to produce disease in a hostorganism. A bacterium may be virulent for one species of host organism(e.g., a mouse) and not virulent for another species of host organism(e.g., a human). Hence, broadly, an “attenuated” bacterium or strain ofbacteria is attenuated in virulence toward at least one species of hostorganism that is susceptible to infection and disease by a virulent formof the bacterium or strain of the bacterium. As used herein, the term“genetic locus” is a broad term and comprises any designated site in thegenome (the total genetic content of an organism) or in a particularnucleotide sequence of a chromosome or replicating nucleic acid molecule(e.g., a plasmid), including but not limited to a gene, nucleotidecoding sequence (for a protein or RNA), operon, regulon, promoter,regulatory site (including transcriptional terminator sites, ribosomebinding sites, transcriptional inhibitor binding sites, transcriptionalactivator binding sites), origin of replication, intercistronic region,and portions therein. A genetic locus may be identified andcharacterized by any of a variety of in vivo and/or in vitro methodsavailable in the art, including but not limited to, conjugation studies,crossover frequencies, transformation analysis, transfection analysis,restriction enzyme mapping protocols, nucleic acid hybridizationanalyses, polymerase chain reaction (PCR) protocols, nuclease protectionassays, and direct nucleic acid sequence analysis. As used herein, theterm “infection” has the meaning generally used and understood bypersons skilled in the art and includes the invasion and multiplicationof a microorganism in or on a host organism (“host”, “individual”,“patient”) with or without a manifestation of a disease (see,“virulence” above). Infectious microorganisms include pathogenicviruses, such as Avian Influenza, that can cause serious diseases wheninfecting an unprotected individual. An infection may occur at one ormore sites in or on an individual. An infection may be unintentional(e.g., unintended ingestion, inhalation, contamination of wounds, etc.)or intentional (e.g., administration of a live vaccine strain,experimental challenge with a pathogenic vaccine strain). In avertebrate host organism, such as a mammal, a site of infectionincludes, but is not limited to, the respiratory system, the alimentarycanal (gut), the circulatory system, the skin, the endocrine system, theneural system, and intercellular spaces. Some degree and form ofreplication or multiplication of an infective microorganism is requiredfor the microorganism to persist at a site of infection. However,replication may vary widely among infecting microorganisms. Accordingly,replication of infecting microorganisms comprises, but is not limitedto, persistent and continuous multiplication of the microorganisms andtransient or temporary maintenance of microorganisms at a specificlocation. Whereas “infection” of a host organism by a pathogenicmicroorganism is undesirable owing to the potential for causing diseasein the host, an “infection” of a host individual with a live vaccinecomprising genetically altered, attenuated Salmonella bacterial strainas described herein is desirable because of the ability of the bacterialstrain to elicit a protective immune response to antigens of AvianInfluenza virus that cause avian influenza in humans and other mammals.

As used herein, the terms “disease” and “disorder” have the meaninggenerally known and understood in the art and comprise any abnormalcondition in the function or well being of a host individual. Adiagnosis of a particular disease or disorder, such as Avian Influenza,by a healthcare professional may be made by direct examination and/orconsideration of results of one or more diagnostic tests.

A “live vaccine composition”, “live vaccine”, “live bacterial vaccine”,and similar terms refer to a composition comprising a strain of liveSalmonella bacteria that expresses at least one antigen of AvianInfluenza, e.g., the H antigen, the N antigen, or a combination thereof,such that when administered to an individual, the bacteria will elicitan immune response in the individual against the Avian Influenzaantigen(s) expressed in the Salmonella bacteria and, thereby, provide atleast partial protective immunity against Avian Influenza. Suchprotective immunity may be evidenced by any of a variety of observableor detectable conditions, including but not limited to, diminution ofone or more disease symptoms (e.g., respiratory distress, fever, pain,diarrhea, bleeding, inflammation of lymph nodes, weakness, malaise),shorter duration of illness, diminution of tissue damage, regenerationof healthy tissue, clearance of pathogenic microorganisms from theindividual, and increased sense of well being by the individual.Although highly desired, it is understood by persons skilled in the artthat no vaccine is expected to induce complete protection from a diseasein every individual that is administered the vaccine or that protectiveimmunity is expected to last throughout the lifetime of an individualwithout periodic “booster” administrations of a vaccine composition. Itis also understood that a live vaccine comprising a bacterium describedherein may be, at the discretion of a healthcare professional,administered to an individual who has not presented symptoms of AvianInfluenza but is considered to be at risk of infection or is known toalready have been exposed to Avian influenza virus, e.g., by proximityor contact with Avian Influenza patients or virally contaminated air,liquids, or surfaces.

The terms “oral”, “enteral”, “enterally”, “orally”, “non-parenteral”,“non-parenterally”, and the like, refer to administration of a compoundor composition to an individual by a route or mode along the alimentarycanal. Examples of “oral” routes of administration of a vaccinecomposition include, without limitation, swallowing liquid or solidforms of a vaccine composition from the mouth, administration of avaccine composition through a nasojejunal or gastrostomy tube,intraduodenal administration of a vaccine composition, and rectaladministration, e.g., using suppositories that release a live bacterialvaccine strain described herein to the lower intestinal tract of thealimentary canal.

The term “recombinant” is used to describe non-naturally altered ormanipulated nucleic acids, cells transformed, electroporated, ortransfected with exogenous nucleic acids, and polypeptides expressednon-naturally, e.g., through manipulation of isolated nucleic acids andtransformation of cells. The term “recombinant” specifically encompassesnucleic acid molecules that have been constructed, at least in part, invitro using genetic engineering techniques, and use of the term“recombinant” as an adjective to describe a molecule, construct, vector,cell, polypeptide, or polynucleotide specifically excludes naturallyexisting forms of such molecules, constructs, vectors, cells,polypeptides, or polynucleotides.

Cassette, or expression cassette is used to describe a nucleic acidsequence comprising (i) a nucleotide sequence encoding a promoter, (ii)a first unique restriction enzyme cleavage site located 5′ of thenucleotide sequence encoding the promoter, and (iii) a second uniquerestriction enzyme cleavage site located 3′ of the nucleotide sequenceencoding the promoter. The cassette may also contain a multiple cloningsite (MCS) and transcriptional terminator within the 5′ and 3′restriction endonuclease cleavage sites. The cassette may also containcloned genes of interest.

As used herein, the term “salmonella” (plural, “salmonellae”) and“Salmonella” refers to a bacterium that is a serovar of Salmonellaenterica. A number of serovars of S. enterica are known. Particularlypreferred Salmonella bacteria useful in the invention are attenuatedstrains of Salmonella enterica serovar Typhimurium (S. typhimurium) andserovar Typhi (S. typhi) as described herein. As used herein, the terms“strain” and “isolate” are synonymous and refer to a particular isolatedbacterium and its genetically identical progeny. Actual examples ofparticular strains of bacteria developed or isolated by human effort areindicated herein by specific letter and numerical designations (e.g.strains Ty21a, CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800, holavax,M01ZH09, VNP20009).

The definitions of other terms used herein are those understood and usedby persons skilled in the art and/or will be evident to persons skilledin the art from usage in the text. This invention provides live vaccinecompositions for protecting against Avian Influenza comprising liveSalmonella enterica serovars that are genetically engineered to expressone or more Avian Influenza antigen polypeptides, such as the H1, H5, H5(H274Y), H7 or H9 and N1, N2 and N7 antigens of Avian Influenza virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a modified ptrc99a plasmid.

FIGS. 2A and B show a plasmid vectors capable of expressing the H5 or N1antigens cytoplasmically.

FIG. 3 shows modified ptrc99a plasmid with unique restriction sitesengineered into the coding sequence of the N1 gene for rapid exchange ofmutations such as the H274Y.

FIG. 4A shows a plasmid vectors expressing the H5 or N1 antigens in asecreted form as fusions with the hlyA protein.

FIG. 4B shows a plasmid vector expressing HlyB and HlyD genes necessaryfor secretion of HlyA and HlyA fusion proteins.

FIG. 5 shows a plasmid vector for expression of an antigen (e.g., H5 orN1) as a ClyA fusion.

FIG. 6 shows a plasmid vector for expression of an antigen (e.g., H5 orN1) as a autotransporter fusion.

FIG. 7 shows a plasmid vector for expression of an antigen (e.g., H5 orN1) as a colicin E3 fusion.

FIG. 8A shows selection of 5′ and 3′ DNA segments for constructing apCVD442 chromosomal integration vector.

8B shows the vector for disrupting chromosomal genes and capable ofintegration of new genes into the chromosome.

FIG. 9 shows a cloning sequence, from A) synthetic gene expressionvector to B) chromosomal localization vector.

FIG. 10 shows a PCR method for determination of IS200 17.7 and 19.9rearrangement/deletion.

FIG. 11 shows a method to achieve the Suwwan deletion in strains lackingthe 17.7 Cs IS200.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon a combination of bacterial vectorand protein expression technology which results in a unique vaccinewhich is rapidly constructed in response to emerging avian influenza andtheir highly pathogenic derivatives. The present invention is directedto the construction bacterially codon optimized avian and humanInfluenza genes and their incorporation into a Salmonella strain fortherapeutic use in the prevention of avian influenza and highlypathogenic derivatives. An antigen-expressing plasmid or chromosomalconstruct in the bacterial strains described herein may also contain oneor more transcriptional terminators adjacent to the 3′ end of aparticular nucleotide sequence on the plasmid to prevent undesiredtranscription into another region of the plasmid or chromosome. Suchtranscription terminators thus serve to prevent transcription fromextending into and potentially interfering with other critical plasmidfunctions, e.g., replication or gene expression. Examples oftranscriptional terminators that may be used in the antigen-expressingplasmids described herein include, but are not limited to, the T1 and T2transcription terminators from 5S ribosomal RNA bacterial genes (see,e.g., FIGS. 1-5; Brosius and Holy, Proc. Natl. Acad. Sci. USA, 81:6929-6933 (1984); Brosius, Gene, 27(2): 161-172 (1984); Orosz et al.,Eur. J. Biochem., 20 (3): 653-659 (1991)).

The mutations in an attenuated bacterial host strain may be generated byintegrating a homologous recombination construct into the chromosome orthe endogenous Salmonella virulence plasmid (Donnenberg and Kaper, 1991;Low et al. (Methods in Molecular Medicine, 2003)). In this system, asuicide plasmid is selected for integration into the chromosome by afirst homologous recombination event, followed by a second homologousrecombination event which results in stable integration into thechromosome. The antigen-expressing chromosomal integration constructsdescribed herein comprise one or more nucleotide sequences that encodeone or more polypeptides that, in turn, comprise one or more AvianInfluenza antigens, such as the hemagglutinin and neuraminidasepolypeptide antigens, or immunogenic portions thereof, from AvianInfluenza virus and highly pathogenic derivatives. Such coding sequencesare operably linked to a promoter of transcription that functions in aSalmonella bacterial strain even when such a bacterial strain isingested, i.e., when a live vaccine composition described herein isadministered orally to an individual. A variety of naturally occurring,recombinant, and semi-synthetic promoters are known to function inenteric bacteria, such as Escherichia coli and serovars of S. enterica(see, e.g., Dunstan et al., Infect. Immun., 67(10): 5133-5141 (1999)).Promoters (P) that are useful in the invention include, but are notlimited to, well known and widely used promoters for gene expressionsuch as the naturally occurring Plac of the lac operon and thesemi-synthetic Ptrc (see, e.g., Amman et al., Gene, 25 (2-3): 167-178(1983)) and Ptac (see, e.g., Aniann et al., Gene, 69(2): 301-315(1988)), as well as PpagC (see, e.g., Hohmann et al., Proc. Natl. Acad.Sci. USA, 92. 2904-2908 (1995)), PpmrH (see, e.g., Gunn et al., Infect.Immun., 68: 6139-6146 (2000)), PpmrD (see, e.g., Roland et al., J.Bacteriol., 176: 3589-3597 (1994)), PompC (see, e.g., Bullifent et al.,Vaccine, 18: 2668-2676 (2000)), PnirB (see, e.g., Chatfield et al.,Biotech. (NY), 10: 888-892 (1992)), PssrA (see, e.g., Lee et al., J.Bacteriol. 182. 771-781 (2000)), PproU (see, e.g., Rajkumari andGowrishankar, J. Bacteriol., 183. 6543-6550 (2001)), Pdps (see, e.g.,Marshall et al., Vaccine, 18: 1298-1306 (2000)), and PssaG (see, e.g.,McKelvie et al., Vaccine, 22: 3243-3255 (2004)), Some promoters areknown to be regulated promoters that require the presence of some kindof activator or inducer molecule in order to transcribe a codingsequence to which they are operably linked. However, some promoters maybe regulated or inducible promoters in E. coli, but function asunregulated promoters in Salmonella. An example of such a promoter isthe well known trc promoter (“Ptrc”, see, e.g., Amman et al., Gene,25(2-3): 167-178 (1983); Pharmacia-Upjohn). As with Plac and Ptac, Ptrcfunctions as an inducible promoter in Escherichia coli (e.g., using theinducer molecule isopropyl-p-D-18 thio-galactopyranoside, “IPTG”),however, in Salmonella bacteria having no LacI repressor, Ptrc is anefficient constitutive promoter that readily transcribes Avian Influenzaantigen-containing polypeptide coding sequences present onantigen-expressing plasmids described herein. Accordingly, such aconstitutive promoter does not depend on the presence of an activator orinducer molecule to express an antigen-containing polypeptide in astrain of Salmonella.

The Avian Influenza antigen-expressing chromosomal integrationconstructs which integrate into the live vaccine strains also contain anorigin of replication (ori) that enables the precursor plasmids to bemaintained as multiple copies in certain the bacterial cells which carrythe lamda pir element. For the process of cloning DNA, a number ofmulti-copy plasmids that replicate in Salmonella bacteria are known inthe art, as are various origins of replications for maintaining multiplecopies of plasmids. Preferred origins of replications for use in themulti-copy antigen-expressing plasmids described herein include theorigin of replication from the multi-copy plasmid pBR322 (“pBR ori”;see, e.g., Maniatis et al., In Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982), pp. 479-487;Watson, Gene, 70: 399-403, 1988), the low copy origin of replicationfrom pACYC177, and the origin of replication of pUC plasmids (“pUCori”), such as found on plasmid pUC 18 (see, e.g., Yanish-Perron et al.,Gene, 33: 103-119 (1985)). Owing to the high degree of genetic identityand homology, any serovar of S. enterica may be used as the bacterialhost for a live vaccine composition for Avian Influenza, provided thenecessary attenuating mutations and antigen-expressing plasmids asdescribed herein are also employed. Accordingly, serovars of S. entericathat may be used in the invention include those selected from the groupconsisting of Salmonella enterica serovar Typhimurium (S. typhimurium),Salmonella montevideo, Salmonella enterica serovar Typhi (S. typhi),Salmonella enterica serovar Paratyphi B (S. paratyphi B), Salmonellaenterica serovar Paratyphi C (S. paratyphi C), Salmonella entericaserovar Hadar (S. hadar), Salmonella enterica serovar Enteriditis (S.enteriditis), Salmonella enterica serovar Kentucky (S. kentucky),Salmonella enterica serovar Infantis (S. infantis), Salmonella entericaserovar Pullorum (S. pullorum), Salmonella enterica serovar Gallinarum(S. gallinarum), Salmonella enterica serovar Muenchen (S. muenchen),Salmonella enterica serovar Anaturn (S. anatum), Salmonella entericaserovar Dublin (S. dublin), Salmonella enterica serovar Derby (S.derby), Salmonella enterica serovar Choleraesuis var. kunzendorf (S.cholerae kunzendorf), and Salmonella enterica serovar minnesota (S.Minnesota).

The vaccine compositions described herein may be administered orally toan individual in any form that permits the Salmonella bacterial strainof the composition to remain alive and to persist in the gut for a timesufficient to elicit an immune response to one or more Avian Influenzaantigens of Avian Influenza virus and highly pathogenic derivativesexpressed in the Salmonella strain. For example, the live bacterialstrains described herein may be administered in relatively simple bufferor saline solutions at physiologically acceptable pH and ion content. By“physiologically acceptable” is meant whatever is compatible with thenormal functioning physiology of an individual who is to receive a livevaccine composition described herein. Preferably, bacterial strainsdescribed herein are suspended in otherwise sterile solutions ofbicarbonate buffers, phosphate buffered saline (PBS), or physiologicalsaline, that can be easily swallowed by most individuals. However,“oral” routes of administration may include not only swallowing from themouth a liquid suspension or solid form comprising a live bacterialstrain described herein, but also administration of a suspension of abacterial strain through a nasojejunal or gastrostomy tube, and rectaladministration, e.g., by using a suppository comprising a live bacterialstrain described herein to establish an infection by such bacterialstrain in the lower intestinal tract of the alimentary canal.Accordingly, any of a variety of alternative modes and means may beemployed to administer a vaccine composition described herein to thealimentary canal of an individual if the individual cannot swallow fromthe mouth.

In a preferred embodiment of the invention, the bacteria have geneticmodifications which result in the expression of at least onehemagglutinin and one neuraminidase, where each gene is optimized forbacterial expression in at least one codon. In a most preferredembodiment, the hemagglutinin and neuraminidase genes are furthermodified to be secreted by the bacteria as heterologous fusion proteins.In a most preferred embodiment, the neuraminidase and hemagglutininheterologous fusion proteins are integrated into the chromosome in deltaIS200 sites.

In a preferred embodiment, the bacterial strains are geneticallystabilized by deletion of IS200 elements, which reduces their geneticrecombination potential.

In another embodiment, the bacterial strains are genetically stabilizedby deletion of phage and prophage elements, which reduces their geneticrecombination and transduction potential.

In another embodiment, the bacterial strains are genetically isolatedfrom phage infection by constitutive expression of the P22 C2 repressor,which reduces their ability to be infected by phage and the subsequenttransduction of genes by such phage.

In another embodiment, the bacterial strains have genetically definedflagellar antigens, or no flagellar antigens, which reduces the immunesystem elimination of the vector, enhancing its immunization potentialin second immunizations.

In a preferred embodiment, the genetically modified bacteria are used inanimals, including humans, birds, dogs and pigs, for protection againstavian influenza and highly pathogenic derivatives.

In another embodiment, a kit allows for rapid construction of abacterial vaccine which is closely matched to an emerging AvianInfluenza or its highly pathogenic derivative.

FIG. 1 shows a modified ptrc99a plasmid. The SphI site within themultiple cloning site has been deleted making the upstream SphI siteunique and useful for subcloning into pCVD vectors. In addition, NotIand PacI sites are added downstream of the t₁t₂ terminators also for usein subcloning into pCVD vectors.

FIGS. 2A and B show a plasmid vectors capable of expressing the H5 or N1antigens cytoplasmically. “Ptrc” refers to a functional trc promoteroperably linked to a structural coding sequence for an H5 antigen fusionpolypeptide. “T1 T2” refers to the T1 and T2 transcriptional terminatorsof the 5S bacterial ribosomal RNA gene. “bla” refers to thebeta-lactamase gene for ampicillin and carbenicillin resistance. Arrowsindicate direction of transcription. See text for details.

FIG. 3 shows modified ptrc99a plasmid with unique restriction sitesengineered into the coding sequence of the N1 gene for rapid exchange ofmutations such as the H274Y.

FIG. 4A shows a plasmid vectors expressing an antigen (H5 or N1) in asecreted form as fusions with the hlyA protein. Numbers after names ofrestriction endonucleases indicate specific restriction sites in theplasmid. “Ptrc” refers to a functional trc promoter operably linked to astructural coding sequence for an antigen fusion polypeptide. “ColE1ori” refers to the colicin E1 origin of replication. 4B shows thehemolysin secretion HlyB and HlyD proteins in a plasmid vector with adifferent origin of replication, the “M15ori”, which refers the M15origin of replication. See text for details.

FIG. 5 shows ClyA fusion. A plasmid vector for expression of an antigen(e.g., H5 or N1) as a ClyA fusion is shown. The modified trc99a vectorof FIG. 1 is used as a cloning and expression vector for a ClyA:antigenfusion.

FIG. 6 shows Autotransporter fusion. A plasmid vector for expression ofan antigen (e.g., H5 or N1) as a autotransporter (translocator) fusionis shown. The modified trc99a vector of FIG. 1 is used as a cloning andexpression vector for the autotransporter:antigen fusion. “S” refers toa hydrophobic signal sequence.

FIG. 7 shows Colicin E3 (ColE3) fusion. A plasmid vector for expressionof an antigen (e.g., H5 or N1) as a colicin E3 fusion is shown. Themodified trc99a vector of FIG. 1 is used as a cloning and expressionvector for the ColE3:antigen fusion.

FIG. 8 shows pCVD knockout constructs. A. Selection of 5′ and 3′ DNAsegments for constructing a pCVD442 chromosomal integration vector fordisrupting chromosomal genes and integration of new genes into thechromosome. The 5′ and 3′ segments may be selected completely within thegene (a), partly within and partly outside (b) or completely outside (c)or any combination of the above so long as in each case there is a gapof at least one nucleotide such that the recombination event results insuch gap introduced into the gene as a deletion resulting ininactivation of the gene. When a foreign gene is inserted such as inFIG. 8B, then the inserted gene also results in a gene disruptionfollowing integration and resolution. B. A localization vector with 5′and 3′ sequence flanking a multiple-cloning sites (SphI/NotI) into whichan expression cassette containing a gene of interest (e.g., an antigensuch as H5 or N1, or another gene of interest such as the P22 phage C2phage repressor protein).

FIG. 9 shows a cloning sequence, from A) synthetic gene within theexpression vector to B) chromosomal localization vector. First, asynthetic gene is generated using standard molecular techniques, thegene is then cloned into an expression vector and then subcloned into apCVD vector for chromosomal localization. “H274Y” refers to thehistidine to tyrosine mutation that confers oseltamivir resistance.

FIG. 10 shows a determination of IS200 17.7 and 19.9rearrangement/deletion. The Suwwan deletion is a recombination eventbetween two IS200 elements located at 17.7 and 19.9 Cs. Ifoligonucleotide primers are generated (P1; forward) to unique sequencesbefore the 17.7 and (P2; reverse) after the 19.9, no PCR product will begenerated under standard short PCR conditions (typically 500 bp to10,000 bp) because the distance between the two points is too long(greater than 20,000 bp). However, following a Suwwan deletion, the twopoints are in relative close proximity and a PCR product will readily begenerated.

FIG. 11 shows a method to generate strains capable of undergoing theSuwwan deletion in strains lacking the 17.7 Cs IS200. In strains whichlack the 17.7 Cs IS200, an IS200 can be introduced in order to generatethe potential to undergo such a deletion. As depicted in FIG. 8, achromosomal localization vector derived from pCVD442 can be generatedwith cloning sites (SphI/NotI) which will accommodate foreign DNA. Inorder to insert an IS200 in the DNA sequence in the homologous locationto that of Salmonella typhimurium ATCC 14028 is identified (A) betweenthe genes ybjL (1) and ybjM (2) and the DNA flanking that region iscloned as 5′ and 3′ regions into pCVD442 together with the SphI and NotIcloning sites (B) and the IS200 from ATCC 14028 is cloned in between the5′ and 3′ regions (C). Recombination with the chromosome results in theinsertion of the IS200 at the appropriate location (D) allowing for thepotential to spontaneously recombine as shown in FIG. 10.

1.1 Cloning Avian Influenza Antigens for Bacterial Expression.

As described in the present invention, Avian Influenza genes can becloned as a codon-optimized synthetic DNA construct and expressed inbacteria including but not limited to Salmonella. Cloning and expressionof the avian influenza genes uses standard molecular techniques(Sambrook et al., Molecular Cloning, Cold Spring Harbor LaboratoryPress, 1989) and conventional bacterial expression plasmids such aspTrc99a (Pharmacia-Upjohn). This results in a plasmid-based, cytosolicexpression of the antigen. For an example, see Section 2.1. The AvianInfluenza antigens can be further modified for secretion as heterologousfusions. Such fusions can be with previously described for hlyA, clyA,SPATE autotransporter proteins or a novel composition of a fusion withcolicin E3 (colE3). For example, see Section 2.10. These cytosolic andsecreted constructs can be further modified by integration into thebacterial chromosome using standard techniques of targeted homologousrecombination (Donnenberg and Kaper, 1991) where the bacterialexpression cassette is inserted in between the 5′ and 3′ flankingsequences as further described below.

1.2 Improvement of Genetic Stability

Bacterial strains such as Salmonella contain a variety of phage andprophage elements. Activation of such phage elements can result ingenetic rearrangements and/or liberate such phage as Gifsy and Felswhich are capable of transducing other bacterial strains. Such phageelements are known by DNA sequence of entire genomes. If the genomesequence is unknown, such elements may be determined by low stringencyDNA:DNA hybridization. In the present invention, DNA sequencesassociated with phage and prophage elements are disrupted to improvegenetic stability and reduce the potential for transduction. Geneticstability is improved by deletion of IS200 and phage/prophage elements.Deletion of IS200 and phage/prophage elements on the bacterialchromosome is accomplished using standard techniques of targetedhomologous recombination (Donnenberg and Kaper, 1991) where 5′ and 3′flanking sequences of the deletion target (IS200 or phage/prophageelements) are cloned into the pCVD442 vector.

Improvement of genetic stability can be determined by assay ofphenotypic or genotypic properties such as spontaneous rearrangement ofIS200 elements resulting in chlorate resistance (Murray et al., 2004).The ability to rearrange IS200 elements and cause a spontaneous deletionmay be determined by assay of spontaneous chlorate resistant bacterialcolonies on LB media containing chlorate. These colonies are thensubjected to PCR analysis of the genome, combined with DNA sequencing,which is thus definitive in respect to a particular IS200-based deletionin the 17.7 to 19.9 Cs region (See FIG. 8). This and other DNArearrangements, duplications and deletions are also determined bypulse-field gel electrophoresis which compares the DNA banding patternof the parent strain (control strain) to strains in which rearrangementsare to be determined (test strains).

1.3 Genetic Isolation of the Bacterial Vectors from Phage

The bacterial strains which vector the avian influenza antigens can bealtered to genetically isolate them from phage. Genetic isolation isaccomplished by limiting the phage integration through constitutiveexpression of the P22 phage repressor. When exogenous phage enter therepressor inhibits their integration into the chromosome. Under certaincircumstances, the repressor may be proteolyticly cleaved by the RecAprotein. This may be circumvented by eliminating the RecA proteincleavage site through site-directed mutagenesis (Sambrook et al.,Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). The P22repressor is cloned into a bacterial expression vector, such as thetrc99a vector and results in constitutive expression. The expressioncassette may be further modified to be integrated into the chromosomeusing standard techniques of targeted homologous recombination(Donnenberg and Kaper, 1991) where the trc99a expression cassette iscloned between the 5′ and 3′ flanking sequences of a deletion target(e.g., IS200 or phage/prophage elements) within a pCVD442 vector.Genetic isolation is tested by experimental infection with phage towhich the bacteria are normally susceptible. Successful construction ofa genetically isolated strain is recognized by substantially lowerinfection rates (e.g., 10 fold lower or more) compared to the parentstrain, where infection rates are determined by plaque forming units(PFU) of phage, such as P22 phage. Moreover, the transduction potentialof such bacteria is also assayed using standard techniques know to thoseskilled in the arts, such as the comparison of transducing potential fora metabolic gene (e.g., purl) from the parent strain compared to themodified strain to an identical recipient strain deficient in the samemetabolic gene (e.g., delta purl). The genetically isolated strainsshows substantially lower (e.g., 10 fold lower or more) ability to havea representative gene transduced to another strain compared to theparent strain.

1.4 Construction of Bacteria with Genetically Defined Flagellar (fH)Antigens or no fH antigens.

In another embodiment, the bacterial strains have genetically definedflagellar antigens, or no flagellar antigens, which reduces the immunesystem elimination of the vector, enhancing its immunization potentialin second immunizations. Strains with defined flagellar antigens areconstructed by first selecting substrains that express either the fH1 orfH2 antigens which the bacteria spontaneously generate by inversion of aportion of the gene mediated by the hin recombinase. To select strainsexpressing either fH1 or fH2, the bacteria are plated to standard growthmedia and subjected to a colony lift using nitrocellulose or equivilantmembrane binding matrix, followed by lysis and blocking of the membrane.fH1 and fH2 are selected using fH1 and fH2 antibodies. The correspondingclone is then purified. These clones are further subjected to deletionof the hin recombinase gene using standard homologous recombinationtechniques including lamda red recombinase or pCVD vectors specific fordisrupting hin, thus fixing their flagellar antigen expression.Furthermore, strains without any flagellar antigens may be constructedby deletion of the fliBC genes using standard homologous recombination.These genetically altered strains with stable expression of either fH1,fH2 or no flagellar antigens (fH0) have reduced elimination by theimmune system when they are used for second immunizations where thefirst immunization is a bacterial strain with a different flagellarantigen or no flagellar antigen or where the first immunization is anon-bacterial vaccine including an egg-based vaccine.

1.5 Use of Genetically Modified Bacteria for Protection Against AvianInfluenza and Highly Pathogenic Derivatives.

As described in the present invention, the bacterial strains whichvector the H and N antigens of Avian Influenza and highly pathogenicderivatives are useful as vaccines, resulting protection againstinfection by Influenza strains.

1.6 A Kit for Rapidly Producing Genetically Modified Bacteria forProtection Against Avian Influenza and Highly Pathogenic Derivatives.

A kit according to one embodiment of the invention comprises 1) abacterial strain, 2) pTrc99a expression vectors containing A)neuraminidase and B) hemagglutinin antigens with unique restrictionendonuclease enzymes within the sequence which allows rapid exchange ofsmall segments (such as the N1 amino acid 274) and 3) multiple uniquechromosomal localization vectors targeting a variety of genes includingIS200s, phage elements (especially Gifsy and Fels) and metabolic genes(such as purl, AroA, etc) for insertion of the pTrc99a expressioncassettes with the modified H and N antigens.

In order to more fully illustrate the invention, the followingnon-limiting examples are provided.

Examples of Bacterial Expression of H and N Antigens and Incorporationin Genetically Stabilized and Isolated Strains with Defined FlagellarAntigens and their Use in Protection Against Avian Influenza and HighlyPathogenic Derivatives.

2.1 Example of Methods for Obtaining Bacterial Strains of theAppropriate Genetic Background.

Bacterial strains useful in the invention include strains of knownsafety when administered to humans including but not limited to Ty21a,CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800, holavax, M01ZH09,VNP20009. These strains contain defined mutations within specificserotypes of bacteria. The invention also includes the use of these samemutational combinations contained within alternate serotypes or strains.Each of the mutations can be generated by chromosomal deletiontechniques known to those skilled in the arts. Generally, the mutationalcombination includes at least two mutations. Such mutations are madesequentially and generally involve the elimination of antibioticresistance markers. The process therefore consists of a first step inselection of an appropriate serotype based upon the known speciesspecificity (e.g, S. typhi is human specific and S. typhimurium hasbroad species specificity including humans, birds, pigs and many othervertebrates). Thus, if the target species for immunization were limitedto humans, S. typhi would be appropriate. If more species are desired tobe immunized including humans, birds, pigs, dogs, horses and many othervertebrates, then other serotypes may be used. In a preferredembodiment, as S. typhimurium and S. montevidio which havenon-overlapping O-antigen presentation (e.g., S. typhimurium is O-1, 4,5, 12 and S. typhi is Vi, S. montevideo is O-6, 7) may be used. Thus, S.typhimurium is a suitable serotype for a prime/boost strategy where S.typhimurium is either the primary vaccine, or the booster vaccine wherethe primary vaccine is another serotype such as S. typhi or S.montivideo. Furthermore, S. typhimurium is suitable for humans, pigs orbirds. A second step follows serotype selection where the first geneticmutation is introduced which may involve the use of antibioticresistance markers and where any antibiotic resistance makers are theneliminated, followed by a third step where a second genetic mutation isintroduced which may involve the use of antibiotic resistance markersand where any antibiotic resistance makers are then also eliminated.Reiteration of genetic deletion and antibiotic marker elimination can beused to supply additional mutations. Methods for reiterative chromosomaldeletion and elimination of antibiotic resistance markers are known tothose skilled in the arts, including TN10 transposon deletion followedby Bochner selection for elimination of the tetracycline antibioticresistance marker, lamda red recombinase deletion followed by fliprecombinase elimination of the antibiotic resistance marker, and suicidevectors such as those containing sucrase gene (e.g., pCVD442, Donnenbergand Kaper, 1991). By way of example, the pCVD442 vector is used in thefollowing manner to create specific genetic deletions. First, thedesired bacterial serotype is selected, such as Salmonella typhimurium.Second, the desired genetic background to be utilized is selected, suchas AroA-, AroD-, htrA- which has been shown to be a safe mutationalcombination. The genes are then deleted in sequence using the pCVD442vector as described by Donnenberg and Kaper 1991. The construction ofthe deletion vector uses DNA sequence for the gene of interest and/orthe flanking 5′ and 3′ DNA. Such DNA may be known and previouslydeposited in a database, or new sequence obtained by methods known tothose skilled in the arts such as low stringency hybridization. Theisolation genes such as AroA, AroD, htrA or any other known attenuatingmutation from Salmonella serotypes where the DNA sequence is not knownis accomplished by low-stringency DNA/DNA hybridization of a Salmonellagenomic DNA library carried in either E. coli or Salmonella LT2 5010(e.g., Sambrook et al., 1989 Molecular Cloning: A laboratory manual(2^(nd) Ed.), Cold Spring Harbor Laboratory Press; Low et al., 1999Nature Biotechnology). A probe for the desired gene such as AroA, AroD,htrA or any other known attenuating mutation is generated from a knownhomologous gene and its corresponding DNA sequence of such as AroA,AroD, htrA or any other known attenuating mutation respectively, by PCR.This fragment is labeled using ³²P-dCTP and used to probe the Salmonellalibrary at low-stringency conditions consisting of 6× sodiumchloride/sodium citrate (SSC), 0.1% sodium dodecylsulfate (SDS),2×Denhardts, 0.5% non-fat dry milk overnight at 55° C. Those skilled inthe art will understand that higher or lower stringency can be achievedusing variation in the 55° C. (higher temperature is a higher stringencyused when excessive binding occurs) combined with higher or lower SSCconcentration (lower buffer concentration is higher stringency used whenexcessive binding occurs; higher concentration is lower stringency usedwhen insufficient binding occurs to generate a signal). Stronglyhybridizing colonies are purified, and plasmids extracted and subjectedDNA sequencing. DNA sequence flanking novel homologue is used togenerate the 5′ and 3′ regions of a sucrase vector which can then beused to specifically delete that genetic element.

Flanking DNA representing 5′ and 3′ regions is then cloned into thesucrase vector using standard techniques such that the unification ofthese regions represents a genetic deletion within the desired gene ofat least one nucleotide. Preferably, most or all of entire gene isdeleted (See FIG. 8). The vector is transformed to the desired strainand selected for antibiotic (ampicillin) resistance. The ampicillinresistance is then eliminated by selection of deletion of the sucrasegene by plating the bacteria to agar plates containing sucrase asdescribed by Donnenberg and Kaper, 1991. Reiteration of these stepstargeted at additional genes results in multiple mutations within thedesired genetic background.

2.2 Example of Generation of Novel Mutational Combinations.

Strains useful in the invention also include novel combinations ofmutations including phoP, phoQ, cdt, cya, crp, poxA, rpoS, htrA, nuoG,pmi, pabA, pts, damA, purA, purB, purI, zwf, aroA, aroC, aroD, gua,cadA, rfc, rjb, rfa, ompR, msbB and the Suwwan deletion. Novelcombinations are selected by experimental analysis of two factors,attenuation and immunogenicity. Attenuation, where the LD₅₀ byadministration in normal immunocompetent mice (e.g., CD1) is greaterthan 10⁵, but not more than 10⁹, and/or the LD₅₀ by IV injection is morethan 10⁴ but not more than 10⁸ is desirable, since this is expected totranslate into a dose in humans which will neither be too potent andprone to potential overdosing and/or side effects, nor over-attenuatedwhich would result in use of very large doses necessitating vastlygreater manufacturing capability. A safe dose (LD₀) is first determinedin mice, and may be extrapolated to other species on a per weight basisor on a basis of surface area (e.g., meters²). A safe dose is thenon-lethal dose determined by a toxicity study using standard methods(Welkos and O'Brian Taylor et al., Proc. Natl. Acad. Sci. USA 84:2833-2837). In non-experimental animals including humans, a dose 1:100or 1:1000 of the LD₀ may first be tested and then escalated to a maximumtolerated dose (MTD) defined as the maximum dose having acceptabletoxicities which are not life threatening. A dose below or up to the MTDmay be used. Immunogenicity is determined by methods know to thoseskilled in the art including wild type strain challenge and/or analysisfor immune response to specific antigens, e.g., ELISA for LPS (e.g.,FLOCKTYPE® Labor Diagnostik, Leipzig, Germany) or to the geneticallyengineered antigens as described further in examples 2.15 and 2.16.Strains which fall into the attenuation range and have the comparativelyhighest immune response as determined by ELISA and wild type immunechallenge using methods known to those skilled in the arts arepreferred. By way of example, the following three combinations aregenerated 1) aroA and purl, 2) aroA and Suwwan, and 3) aroA, purl andSuwwan are generated. DNA sequences for aroA and purl are known for S.typhimurium. The Suwwan deletion is described by Murray et al., 2004.The Suwwan deletion is selected for in ATCC strain 14028 using agarplates containing chlorate. Approximately one in three resistant stainscontain the Suwwan deletion, which is confirmed by PCR using primersoutside of the two IS200 elements (FIG. 8). The introduction of theSuwwan deletion is not followed by restoring antibiotic sensitivity,since chlorate is not clinically relevant and there is no antibioticresistance gene inserted in the process. Thus, using the methodsdescribed by Donnenberg and Kaper 1991 as described in Example 2.1 andthe derivation of the Suwwan deletion, the combined three mutations aregenerated: 1) aroA and purl, 2) aroA and Suwwan, and 3) aroA, purl andSuwwan. These combinations are then determined for LD₅₀ using standardmethods (Welkos and O'Brian Taylor et al., Proc. Natl. Acad. Sci. USA84: 2833-2837) and those with the desired attenuation profile describedabove are selected for further analysis. In a wild type challengeexperiment, the mice are first administered the individual bacterialstrain orally at a safe dose (i.e., an LD₀ or less than the LD₁₀ asdefined from the same LD₅₀ experiment previously performed). Sub-lethaldoses of the attenuated strains are expected to immunize the miceagainst the lethal wild type strain. At a suitable time period (forexample, 2 to 6 weeks, 1 to 12 weeks, or 1 to 53 weeks) following asingle administration of a dose less than the LD₁₀, either a boosterdose also less than the LD₁₀ may be administered and staged for twoadditional weeks, or the challenge experiment may be performed. Thechallenge is performed in the form of an oral administration of a lethaldose of the wild type, usually 10 colony forming units (CFU) or greater,and a survival is monitored over time. Strains with the greatestimmunization potential result in immunized mice with the longestsurvival. Immunization can also be determined by immune response toSalmonella antigens, such as the O-antigens, H-antigens or LPS. Adetermination of anti-LPS is performed using a commercially availableELISA kit. Bacterial strains with the appropriate attenuation andhighest level of demonstrated immunization are used for vaccinecarriers.

2.3 Example of Construction of the Suwwan Deletion in Strains Lackingthe 17.7 IS200 Element.

The method for selection of the Suwwan deletion has been described byMurray et al., 2004 for the Salmonella typhimurium strain ATCC 14028.Since other Salmonella strains lack the additional IS200 element at Cs.17.7, they do not undergo this specific chromosomal deletion. Theinvention further provides a method to allow the Suwwan deletion tooccur in other Salmonella strains, by using a sucrase deletion constructas described above which contains the 3′ and 5′ flanking regions whichoccur in other strains, isolated using analogous primers and providing amultiple cloning site. The IS200 Cs 17.7 is then cloned by PCR into themultiple cloning site of the sucrase vector containing the flankingsequence of the empty IS200 site. Subsequent homologous recombinationresults in the addition of the IS200 to the site where it was previouslyabsent. Subsequent selection for the Suwwan deletion is then performed,resulting in a strain with the analogous Cs 17 Cs 19 deletion.

2.4 Example of Construction of Synthetic, Codon Optimized HemagglutininGenes for Bacterial in a Cytosolic Form in Salmonella.

Codon optimized genes generated by reverse translation (a.k.a.,back-translation) of the Avian Influenza genes or their highlypathogenic derivatives using Salmonella optimized codons and thesynthetic gene constructed by annealing overlapping plus and minusstrand oligonucleotides. For cytoplasmic expression, a second codon GCTis added following the ATG start site, the two codons together with anupstream CC constitute the restriction endonuclease site NcoI (CCATGG).Following the final codon TGA, the restriction endonuclease site HindIIIhas been added, thus, a nucleic acid containing this sequence can berestriction digested with NcoI and HindIII and cloned into theNcoI/HindIII sites of the bacterial expression plasmid trc99a(Pharmacia/Upjohn). For convenience, the trc99a vector is modified toremove the sphI and pstI sites and addition of NotI and PacI sites (FIG.1). This allow, for example, directional subcloning of the expressioncassette consisting of the trc promoter and its ribosomal binding site,any given cloned gene within the multiple cloning site, and thedownstream ribosomal RNA termination signals. SphI and PstI are removedfrom trc99a by restriction digestion with PstI and HindIII, agarose gelanalysis and gel purification of the restriction digested plasmid minusthe small DNA seq cleaved by the restriction enzymes, and ligation of asynthetic oligonucleotide

SEQ ID NO: 001 AGCTTGCA.

Clones may be further confirmed by restriction endonuclease analysis orDNA sequencing. The NotI and PacI sites are added by inverse PCR, wherethe primers consist of

INVNOTF1 SEQ ID NO: 0025′-GATCGCGGCCGCTTAATTAACATTCAAATATGTATCCGCTCATGAG- 3′ and INVNOTR1SEQ ID NO: 003 5′-GATCGCGGCCGCGTATTTAGAAAAATAAACAAAAAGAGTTTG-3′

The forward primer introduces the NotI and PacI sites, and the reverseprimer provides a second NotI site. The linear PCR product is thenrestriction digested with NotI and self-ligated, and transformed to E.coli. Confirmation of the correct clones is obtained by restrictionanalysis, where the isolated plasmids now contain NotI and PacI sites orby DNA sequencing.

Bacterial expression is tested by any applicable technique known tothose skilled in the arts such as ELISA or immunoblot. Such plasmid canbe transferred to a suitable Salmonella strain by standardtransformation techniques to comprise a Salmonella strain whichexpresses the H5 antigen cytoplasmically and is capable of eliciting animmune response.

TABLE 1 Salmonella typhimurium LT2 [gbbct]: 4696 CDS's (1477317 codons)fields: [triplet] [frequency: per thousand] ([number]) UUU 23.3 UCU 7.2UAU 17.1 (25288) UGU 4.8 (7154) (34407) (10665) UUC 15.3 UCC 10.1 UAC11.6 (17079) UGC 6.6 (9817) (22562) (14953) UUA 13.2 UCA 6.2 UAA 1.9(2781) UGA 1.0 (1466) (19499) (9186) UUG 12.4 UCG 9.5 UAG 0.3 (452) UGG15.2 (22479) (18352) (14062) CUU 11.8 CCU 7.2 CAU 13.3 (19643) CGU 18.8(27700) (17442) (10564) CUC 10.4 CCC 6.9 CAC 9.6 (14171) CGC 23.3(34474) (15425) (10235) CUA 4.9 CCA 5.8 CAA 12.7 (18796) CGA 3.6 (5268)(7257) (8501) CUG 53.6 CCG 24.7 CAG 31.0 (45726) CGG 6.9 (10266) (79180)(36447) AUU 29.3 ACU 6.7 AAU 17.8 (26263) AGU 7.3 (10831) (43251) (9935)AUC 24.4 ACC 23.3 AAC 20.1 (29752) AGC 17.4 (25762) (36114) (34480) AUA5.3 ACA 5.8 AAA 31.7 (46882) AGA 2.3 (3451) (7886) (8515) AUG 27.4 ACG18.8 AAG 11.3 (16630) AGG 1.6 (2422) (40490) (27756) GUU 15.5 GCU 12.8GAU 31.6 (46740) GGU 17.4 (25643) (22914) (18891) GUC 18.2 GCC 29.1 GAC20.3 (30060) GGC 35.3 (52100) (26821) (42983) GUA 11.4 GCA 13.0 GAA 35.4(52232) GGA 8.7 (12841) (16792) (19160) GUG 25.2 GCG 42.5 GAG 20.7(30586) GGG 12.0 (17784) (37210) (62843) Coding GC 53.36% 1st letter GC59.34% 2nd letter GC 41.20% 3rd letter GC 59.53%

TABLE 2 Salmonella typhi [gbbct]: 397 CDS's (116164 codons) fields:[triplet] [frequency: per thousand] ([number]) UUU 23.8 (2767) UCU 11.8(1372) UAU 18.9 (2192) UGU 6.3 (727) UUC 15.5 (1804) UCC 10.4 (1209) UAC13.2 (1538) UGC 5.6 (652) UUA 15.3 (1783) UCA 14.3 (1656) UAA 1.7 (193)UGA 1.3 (155) UUG 12.3 (1434) UCG 9.6 (1119) UAG 0.4 (49) UGG 12.8(1491) CUU 15.8 (1834) CCU 10.0 (1165) CAU 11.4 (1319) CGU 15.2 (1765)CUC 10.7 (1247) CCC 6.7 (783) CAC 7.2 (839) CGC 12.5 (1456) CUA 6.5(754) CCA 8.7 (1012) CAA 13.9 (1618) CGA 6.3 (729) CUG 35.4 (4110) CCG14.5 (1689) CAG 27.4 (3183) CGG 7.8 (908) AUU 27.7 (3214) ACU 14.2(1647) AAU 26.6 (3086) AGU 12.6 (1467) AUC 20.5 (2382) ACC 20.5 (2377)AAC 22.6 (2629) AGC 16.5 (1921) AUA 9.5 (1107) ACA 13.5 (1568) AAA 35.8(4156) AGA 5.7 (666) AUG 26.1 (3037) ACG 15.9 (1845) AAG 17.1 (1989) AGG4.5 (520) GUU 20.1 (2339) GCU 17.5 (2036) GAU 34.0 (3947) GGU 19.7(2286) GUC 15.6 (1817) GCC 22.0 (2559) GAC 20.1 (2332) GGC 22.5 (2612)GUA 12.8 (1484) GCA 20.5 (2382) GAA 35.1 (4080) GGA 12.2 (1414) GUG 19.6(2274) GCG 21.2 (2461) GAG 21.1 (2446) GGG 13.2 (1532) Coding GC 48.16%1st letter GC 53.73% 2nd letter GC 40.62% 3rd letter GC 50.14%

A codon optimized sequence is generated by reverse or back translation,i.e., the conversion of the amino acid sequence into the appropriate DNAsequence. Because of redundancy of the genetic code, many amino acidshave more than one possible codon set which will translate to theappropriate amino acid. Recognition sequences representations use thestandard abbreviations (Eur. J. Biochem. 150:1-5, 1985) to representambiguity.

-   -   R=G or A    -   Y=C or T    -   M=A or C    -   K=G or T    -   S=G or C    -   W=A or T    -   B=not A (C or G or T)    -   D=not C (A or G or T)    -   H=not G (A or C or T)    -   V=not T (A or C or G)    -   N=A or C or G or T

Based upon the codon usage table which indicates preferences as higherpercentages of usage and therefore optimal codons, a complete sequencecan be back translated.

The H5 hemagglutinin gene has a number of known sequence, see e.g.,Genbank LOCUS NC_(—)007362, isolated from a goose in Guangdong, China in1996, or a more preferably, a recent isolate such as CY019432, obtainedfrom a 26 year old female human infected with avian influenza inIndonesia in 2006, expressly incorporated herein by reference.

The result of the reverse translation of CY019432 into a Salmonellacodon optimized sequence is shown below

(SEQ ID NO: 004) GATCCCATGGCTGAGAAAATTGTGCTGCTGCTGTCCATTGTGTCGCTGGTCAAAAGCGATCAGATCTGCATTGGCTACCATGCGAACAATAGCACCGAACAGGTTGATACCATTATGGAGAAAAACGTCACCGTGACCCATGCGCAGGACATCCTGGAAAAAACCCATAATGGCAAACTGTGCGATCTGGATGGCGTCAAACCGCTGATCCTGAAAGATTGCAGCGTGGCGGGTTGGCTGCTGGGCAACCCGATGTGCGATGAATTTATCAATGTTCCGGAATGGAGCTATATTGTGGAAAAAGCGAATCCGACCAACGATCTGTGTTATCCGGGTTCGTTTAACGATTACGAAGAACTGAAACACCTGCTGAGCCGTATTAATCATTTTGAAAAAATCCAGATTATTCCGAAATCGAGCTGGTCGGACCACGAGGCGAGCTCGGGCGTTTCCTCCGCCTGCCCGTATCTGGGTAGCCCGAGCTTTTTTCGTAATGTGGTCTGGCTGATCAAAAAAAATTCCACGTACCCGACCATTAAAAAAAGCTATAACAACACCAACCAGGAAGATCTGCTGGTGCTGTGGGGCATTCATCATCCGAACAATGAAGAAGAACAGACCCGCCTGTACCAGAATCCGACCACCTATATTAGCATTGGCACCAGCACCCTGAATCAGCGTCTGGTTCCGAAAATTGCGACCCGCAGCAAAGTGAACGGCCAGTCCGGTCGTATGGAATTTTTTTGGACCATTCTGAAACCGAATGATGCCATCAACTTTGAATCCAATGGCAATTTTATCGCGCCGGAATACGCGTATAAAATCGTGAAAAAAGGCGATAGCGCCATTATGAAAAGCGAACTGGAATACTCCAACTGCAATACGAAATGTCAGACGCCGATGGGCGCGATCAACAGCTCGATGCCGTTTCACAACATCCATCCGCTGACCATTGGCGAGTGTCCGAAATATGTCAAAAGCAGCCGCCTGGTGCTGGCCACCGGCCTGCGCAATTCGCCGCAGCGTGAAAGCCGTCGCAAAAAACGTGGCCTGTTTGGCGCGATTGCGGGCTTCATTGAAGGCGGCTGGCAGGGTATGGTCGACGGCTGGTACGGTTATCATCATAGCAACGAACAGGGTAGCGGCTATGCGGCGGATAAAGAATCCACCCAGAAAGCCATCGATGGTGTCACGAATAAAGTGAATAGCATTATTGACAAAATGAACACCCAGTTCGAGGCGGTCGGCCGCGAGTTTAATAATCTGGAACGCCGCATTGAAAATCTGAATAAAAAAATGGAAGATGGCTTTCTGGACGTTTGGACCTATAACGCGGAACTGCTGGTCCTGATGGAGAACGAACGCACGCTGGACTTTCATGATTCCAACGTGAAAAATCTGTACGATAAAGTTCGTCTGCAGCTGCGCGACAATGCCAAAGAACTGGGCAACGGCTGTTTCGAGTTTTATCATAAATGTGATAACGAATGCATGGAATCCATTCGTAACGGTACCTACAACTATCCGCAGTATAGCGAAGAAGCGCGCCTGAAACGTGAAGAGATTTCGGGTGTGAAACTGGAATCCATTGGCACCTATCAGATTCTGTCCATTTATAGCACCGTCGCCAGCTCCCTGGCCCTGGCCATTATGATTGCGGGCCTGAGCCTGTGGATGTGCTCCAACGGCTCCCTGCAGTGTCGCATCTGCATCTGAAAGCTTGATC.

The sequence begins with four spacer codons for restriction digestionand cloning. The Genbank sequence had a second codon inserted (GCT),which is a strong translational second codon in gram negative bacteria.The initiating codon ATG is underlined as well as the stop codon TGAwhich is followed by the nucleotides for the restriction site HindIIIand four spacer codons.

2.5 Example of Construction of Synthetic, Codon Optimized NeuraminidaseGenes for Bacterial in a Cytosolic Form in Salmonella.

Codon optimized N1 orf (SEQ ID NO: 005) is generated by reversetranslation of the Avian Influenza gene using Salmonella optimizedcodons and the synthetic gene constructed by annealing overlapping plusand minus strand oligonucleotides as described in the example above. Forcytoplasmic expression, a second codon GCT encoding alanine is addedfollowing the ATG start site encoding the initiating methionine, the twocodons together with an upstream CC constitute the restrictionendonuclease site NcoI. Further upstream the nucleotides GACT are addedto increase the distance of the restriction site from the end, enhancingthe abiligy of the enzyme to cut close to the end. Following the finalamino acid codon a TGA stop codon, the restriction endonuclease siteHindIII has been added, thus, a nucleic acid containing this sequencecan be restriction digested with NcoI and HindIII and cloned into theNcoI/HindIII sites of the bacterial expression plasmid trc99a(Pharmacia/Upjohn). Bacterial expression is tested by any applicabletechnique known to those skilled in the arts such as ELISA orimmunoblot. Such plasmids can be transferred to a suitable Salmonellastrain by standard transformation techniques to comprise a Salmonellastrain which expresses the H5 antigen cytoplasmically and whenadministered to an animal is capable of eliciting an immune response asdescribed in example 7.15.

The N1 neuraminidase gene has a known sequence, see Genbank LOCUSNC_(—)007361, expressly incorporated herein by reference.

Reverse translation using Salmonella codon preferences results in thefollowing DNA sequence.

(SEQ ID NO: 005) GATC cc ATG (GCT) AAT CCG AAC CAG AAA ATT ATC ACCATT GGC TCT ATT TGC ATG GTG GTA GGG ATC ATTTCC CTG ATG TTA CAG ATC GGC AAC ATT ATCTCG ATC TGG GTG TCC CAT TCT ATT CAG ACCGGC AAC CAG CAT CAG GCC GAA CCG TGC AATCAA AGC ATT ATC ACC TAC GAA AAT AAC ACCTGG GTA AAT CAG ACC TAT GTT AAT ATT TCAAAC ACC AAC TTC CTG ACC GAA AAA GCG GTGGCA AGT GTA ACC CTC GCC GGT AAC AGC TCGCTG TGT CCT ATT TCT GGC TGG GCG GTA CACAGC AAA GAT AAT GGC ATT CGC ATC GGC TCTAAA GGC GAC GTT TTT GTG ATC CGC GAA CCCTTT ATT TCG TGT AGC CAT CTG GAG TGC CGTACC TTT TTC TTG ACC CAG GGG GCG CTG CTTAAC GAT AAG CAT TCG AAT GGC ACG GTT AAAGAT CGC AGT CCG CAC CGC ACG CTG ATG AGCTGC CCA GTG GGG GAG GCC CCA TCC CCA TACAAC TCG CGC TTC GAA TCC GTC GCT TGG AGCGCC AGC GCG TGC CAC GAT GGT ACG TCT TGGCTG ACG ATC GGC ATT AGC GGT CCG GAC AACGGT GCG GTT GCT GTC CTG AAA TAT AAT GGTATT ATC ACG GAC ACC ATT AAA TCG TGG CGCAAC AAT ATC TTA CGG ACC CAG GAG TCA GAATGC GCC TGC GTG AAT GGC TCT TGC TTT ACGGTC ATG ACC GAT GGC CCG AGT AAT GGC CAAGCG TCC TAT AAA ATT TTT AAA ATG GAA AAAGGG AAA GTT GTG AAG TCA GTG GAA CTT AACGCC CCG AAC TAT CAC TAT GAA GAG TGT TCGTGT TAC CCT GAC GCA GGC GAA ATC ACG TGTGTC TGC CGT GAT AAC TGG CAT GGC AGC AACCGC CCG TGG GTG TCC TTT AAC CAG AAT TTGGAA TAT CAG ATC GGC TAT ATT TGT TCT GGGGTC TTC GGC GAT AAC CCG CGT CCT AAT GACGGC ACC GGC AGC TGT GGC CCG GTA TCC CCCAAT GGT GCG TAT GGC GTT AAG GGT TTC AGTTTC AAA TAC GGT AAT GGC GTG TGG ATT GGTCGC ACC AAA TCA ACC AAC TCG CGG TCG GGTTTT GAA ATG ATC TGG GAT CCG AAT GGC TGGACC GGT ACC GAT AGC TCA TTC TCC GTG AAGCAA GAC ATC GTC GCA ATT ACG GAT TGG TCCGGC TAC AGT GGC AGC TTT GTG CAA CAT CCGGAG CTG ACC GGG CTG GAT TGC ATT CGC CCCTGT TTT TGG GTT GAA CTG ATT CGT GGG CGTCCG AAG GAG TCA ACG ATC TGG ACG AGC GGCAGC AGT ATT AGC TTT TGC GGC GTC AAC AGCGAC ACG GTC GGC TGG AGT TGG CCG GAT GACGCG GAG CTC CCT TTT ACC ATT GAT AAA TAG AAGCTT GATC

The sequence is further optimized for bacterial expression by additionof the appropriate restriction sites for cloning. An NcoI site isengineered using the start codon together with second codon GCT and astop codon is added after the final amino acid codon together with anengineered HindIII site and end spacer. Such a synthetically derived DNAsequence can then be cloned into the NcoI/HindIII sites of the bacterialexpression plasmid pTrc99a and transformed into the Salmonella strain toresult in a vaccine strain expressing the viral antigen.

2.6 Example of Construction of Synthetic, Codon Optimized Genes withUnique Restriction Endonuclease Sites for Rapidly Matching an EmergingPathogen.

Oseltamivir-resistant neuraminidase is an example of an antigen with analtered amino acid sequence which could change antigenicity. The abovesynthetic construct in Example 2.5 above which contains restrictionsites is further modified, where the synthetic sequence containsmutations representing resistance to osetlamivir, such as the histadineto tyrosine mutation at amino acid position 274 (H274Y). First thetrc99a N1 expression construct is restriction endonuclease digested withappropriate sequences. A synthetic DNA construct containing the N1sequence bearing the H274Y variation is obtained through syntheticconstruction and ligated into the restriction endonuclease target sitesof the previously prepared gene. The plasmid is transfected into asuitable bacterial vector. Thus, the new construct is more rapidlygenerated and when expressed in the bacterial vector, results in avaccine antigenically matched to the emerging oseltamivir resistantstrain.

2.7 Example of Secretion of Avian Influenza Antigens and HighlyPathogenic Derivatives Using HlyA Fustion.

Avian influenza antigen polypeptides expressed from antigen-expressingplasmids or chromosomal constructs in the vaccine strains describedherein need not be linked to a signal peptide or other peptide formembrane localization or secretion across the cell membrane. However, byway of further example of a preferred embodiment, a nucleotide sequencethat encodes an H5-HlyA fusion polypeptide useful in the invention isknown in the art, and the corresponding encoded H5-HlyA fusionpolypeptide has the corresponding amino acid sequence. Theantigen-expressing plasmids useful in the invention may be engineered toexpress an Avian Influenza antigen polypeptide intracellularly in a hostSalmonella strain. Preferably, antigen-expressing plasmids orchromosomal expression constructs useful in the invention are engineeredto express secreted forms of Avian Influenza antigen polypeptideextracellularly. Accordingly, Avian Influenza antigen polypeptidesexpressed from antigen-expressing plasmids in the vaccine strainsdescribed herein, are preferably linked to a signal peptide or otherpeptide for membrane localization or secretion across the cell membrane.

Construction of hemolysin A (hlyA) fusions with H5 nucleotide sequenceto result in an hlyA secreted fusion peptide. HlyA fusions are generatedusing plasmids that provide the 60 C terminal amino acids of HLYA[(Gentschev, et al., 1994. Synthesis and secretion of bacterial antigensby attenuated Salmonella via the Escherichia coli hemolysin secretionsystem. Behring Inst. Mitt. 95:57-66; Holland et al. U.S. Pat. No.5,143,830) by methods known to those skilled in the arts and ligatedinto the hlyA fusion vector to generate a nucleic acid sequence encodingan H5::HLYA fusion peptide. The fusion may also be generated as acompletely synthetic DNA construct as described for the hemagglutininand neuraminidase genes.

An example of the CY019432 codon optimized H5 gene operably fused to the60 C-terminal amino acids of HlyA is shown below.

(SEQ ID NO: 006) GATCCCATGGCTGAGAAAATTGTGCTGCTGCTGTCCATTGTGTCGCTGGTCAAAAGCGATCAGATCTGCATTGGCTACCATGCGAACAATAGCACCGAACAGGTTGATACCATTATGGAGAAAAACGTCACCGTGACCCATGCGCAGGACATCCTGGAAAAAACCCATAATGGCAAACTGTGCGATCTGGATGGCGTCAAACCGCTGATCCTGAAAGATTGCAGCGTGGCGGGTTGGCTGCTGGGCAACCCGATGTGCGATGAATTTATCAATGTTCCGGAATGGAGCTATATTGTGGAAAAAGCGAATCCGACCAACGATCTGTGTTATCCGGGTTCGTTTAACGATTACGAAGAACTGAAACACCTGCTGAGCCGTATTAATCATTTTGAAAAAATCCAGATTATTCCGAAATCGAGCTGGTCGGACCACGAGGCGAGCTCGGGCGTTTCCTCCGCCTGCCCGTATCTGGGTAGCCCGAGCTTTTTTCGTAATGTGGTCTGGCTGATCAAAAAAAATTCCACGTACCCGACCATTAAAAAAAGCTATAACAACACCAACCAGGAAGATCTGCTGGTGCTGTGGGGCATTCATCATCCGAACAATGAAGAAGAACAGACCCGCCTGTACCAGAATCCGACCACCTATATTAGCATTGGCACCAGCACCCTGAATCAGCGTCTGGTTCCGAAAATTGCGACCCGCAGCAAAGTGAACGGCCAGTCCGGTCGTATGGAATTTTTTTGGACCATTCTGAAACCGAATGATGCCATCAACTTTGAATCCAATGGCAATTTTATCGCGCCGGAATACGCGTATAAAATCGTGAAAAAAGGCGATAGCGCCATTATGAAAAGCGAACTGGAATACTCCAACTGCAATACGAAATGTCAGACGCCGATGGGCGCGATCAACAGCTCGATGCCGTTTCACAACATCCATCCGCTGACCATTGGCGAGTGTCCGAAATATGTCAAAAGCAGCCGCCTGGTGCTGGCCACCGGCCTGCGCAATTCGCCGCAGCGTGAAAGCCGTCGCAAAAAACGTGGCCTGTTTGGCGCGATTGCGGGCTTCATTGAAGGCGGCTGGCAGGGTATGGTCGACGGCTGGTACGGTTATCATCATAGCAACGAACAGGGTAGCGGCTATGCGGCGGATAAAGAATCCACCCAGAAAGCCATCGATGGTGTCACGAATAAAGTGAATAGCATTATTGACAAAATGAACACCCAGTTCGAGGCGGTCGGCCGCGAGTTTAATAATCTGGAACGCCGCATTGAAAATCTGAATAAAAAAATGGAAGATGGCTTTCTGGACGTTTGGACCTATAACGCGGAACTGCTGGTCCTGATGGAGAACGAACGCACGCTGGACTTTCATGATTCCAACGTGAAAAATCTGTACGATAAAGTTCGTCTGCAGCTGCGCGACAATGCCAAAGAACTGGGCAACGGCTGTTTCGAGTTTTATCATAAATGTGATAACGAATGCATGGAATCCATTCGTAACGGTACCTACAACTATCCGCAGTATAGCGAAGAAGCGCGCCTGAAACGTGAAGAGATTTCGGGTGTGAAACTGGAATCCATTGGCACCTATCAGATTCTGTCCATTTATAGCACCGTCGCCAGCTCCCTGGCCCTGGCCATTATGATTGCGGGCCTGAGCCTGTGGATGTGCTCCAACGGCTCCCTGCAGTGTCGCATCTGCATCCCCGGGTCAACTTATGGGAGCCAGGACTATCTTAATCCATTGATTAATGAAATCAGCAAAATCATTTCAGCTGCAGGTAATTTGGATGTTAAGGAGGAAAGATCTGCCGCTTCTTTATTGCAGTTGTCCGGTAATGCCAGTGATTTTTCATATGGACGGAACTCAATAACTTTGACAGCATCAGC ATAAAAGCTTGATC

The sequence begins with four spacer codons for restriction digestionand cloning. The Genbank sequence had a second codon inserted (GCT) inthe H5 gene, which is a strong translational second codon in gramnegative bacteria. The initiating codon ATG is underlined. A SmaIrestriction endonuclease site has been added in place of the H5 stopcodon to facilitate cloning and the fusion of the peptides, followed byin-frame coding sequence for the 60 C-terminal amino acids of the HlyAgene, which ends with the stop codon TAA (underlined) which is followedby the nucleotides for the restriction site HindIII and four spacercodons. A naturally occurring PacI restriction endonuclease siteoccurring within HlyA has been conservatively altered to facilitate thepotential use of PacI as a restriction site outside of the codingsequence.

The secretion of the hlyA fusion requires the presence of the HlyBD geneproducts. In order to provide for the presence of the HlyBD genes, aplasmid containing the genes may be used (FIG. 4), or preferably, theHlyBD genes are cloned within a sucrase vector such as an IS200 phagerecombinase, flagellar, or hin pCVD deletion vector. The entire exportcassette can be excised from pVDL9.3 as a NotI-digested fragment andcloned into the Not1 site of a sucrase vector, which when recombinedwith the chromosome, results in deletion of the IS200 phage recombinase,flagellar, or hin and insertion of the HlyBD genes into the chromosome.

2.8 Example of Secretion of Avian Influenza Antigens and HighlyPathogenic Derivatives Using ClyA Fusion.

Construction of clyA fusions with hemagglutinin and neuraminidaseantigens are generate according to the methods of Galen et al. (2004Infection and Immunity 72: 7096-7106).

2.9 Example of Secretion of Avian Influenza Antigens and HighlyPathogenic Derivatives Using Autotransporter Fusions.

Construction of autotransporter fusions with hemagglutinin andneuraminidase antigens. Autotransporter chimeric proteins are capable ofself-transportation/secretion outside the bacterial cell. Hemagglutininand neuraminidase fusions with the IgA protease autotransported proteinof Nisseria gonorrhoeae are constructed according to the methods ofVeiga et al., 2003 J. Virol. 2003 77: 13396-13398) and Oomen et al.,2004 EMBO Journal 23: 1257-1266. The resulting fusion construct, whentransfected into a bacterial vector, results in a vaccine strain whichsecretes the neuraminidase and hemagglutinin antigens.

2.10 Example of Secretion of Avian Influenza Antigens and HighlyPathogenic Derivatives Using Colicin E3 Fusions.

Colicin E3 (ColE3) is a bacterial ribosomal RNA inactivating toxin.ColE3 is neutralized within the cells that express it by an antitoxinwhich inhibits is anti-ribosomal activity. An inactivated ColE3 iscloned from a colE3 containing bacterial strain (e.g., ColE3-CA38). PCRprimers consist of a forward primer which clones the start codon with asecond added codon and providing an NcoI cloning site and a reverseprimer which contains a SmaI (blunt end) cloning site. The PCR primer issituated sufficiently far down the sequence, such that the C-terminalportion of the protein is absent, thus inactivating the toxic activitywhile retaining the secretion activity. The hemagglutinin andneuraminidase antigens are cut with NcoI and HindIII, blunt end polishedand ligated in-frame into the SmaI site of the truncated ColE3 protein.The DNA orientation is then confirmed by restriction analysis and DNAsequencing. When transformed into the bacterial vector, the DNAconstruct results in secreted hemagglutinin or neuraminidase antigens.

2.11 Example of Genetic Stabilization by Deletion of IS200 Elements.

Using the generalized pCVD442 method homologous recombination techniqueusing the vector pCVD442 (Donnenberg and Kaper, 1991), IS200 elementscan be deleted. Such elements in the Salmonella typhimurium strain LT2includes LOCUS NC_(—)003197, having a sequence well known in the art.The IS200 elements contain a transposase with a well known amino acidsequence.

Additional IS200 elements, if not known by DNA sequence, can be isolatedby low stringency hybridization. The isolation IS200 elements fromSalmonella by low-stringency DNA/DNA hybridization of a Salmonellagenomic DNA library carried in Salmonella LT2 5010 (e.g., Low et al.,1999 Nature Biotechnology). A probe for IS200 is generated from a knownIS200 element by PCR. This fragment is labeled using ³²P-dCTP and usedto probe the Salmonella library at low-stringency conditions consistingof 6× sodium chloride/sodium citrate (SSC), 0.1% sodium dodecylsulfate(SDS), 2×Denhardts, 0.5% non-fat dry milk overnight at 55° C. Stronglyhybridizing colonies are purified, and plasmids extracted and subjectedDNA sequencing. DNA sequence flanking novel IS200 elements is used togenerate the 5′ and 3′ regions of a sucrase vector which can then beused to specifically delete that IS200 element.

By way of specific example, the IS200 located in 17.7 Cs. can be deletedusing a 5′ section generated using the PCR primers

2415F1 (IS200 5′F with SacI) SEQ ID NO: 007GATCGAGCTCGGCTTAATTATTGCCCAGCTTGCGCTGG and2415R1 (IS200 5′R with poly linker) SEQ ID NO: 008CCCCGCATGCGGGGCTCGAGGGGGCCATATAGGCCGGGGATTTAAATGGGGCGGCCGCAAAAAAAATCCTGGCGCAGGGCCAGG and a 3′section using the primers 2413F1 (IS200 3′F with poly) SEQ ID NO: 009CCCCGCATGCGGGGAGATCTGGGGTTAATTAAGGGGTCTAGAGGGGGCGGCCGCAGGACTATATTTAGGGCGAAACAGC and 2413R1 (IS200 3′R with SalI)SEQ ID NO: 010 GATCGTCGACGACTAAACATGATTCCAACAATCACG.

The 5′ section is cloned into the pCVD442 vector using Sac1 and SphI,and subsequently, after isolation and identification of the appropriateclone, the 3′ section is added using the restriction endonucleaseenzymes SphI and SalI. The primers also provide a multiple cloning sitecontaining Not1, Pac1, BstY1, SphI, SfiI, Swa1, which can be used todeliver exogenous genes such as the H5 and N1, the lamda repressor C1,or the hlyBD (protein channel) described further below.

2.12 Example of Genetic Stabilization by Deletion of Phage Elements.

Bacterial strains containing phage or prophage elements may have thephage enter a lytic cycle in which they may undergo recombinationinversion. Bacterial strains such as Salmonella contain Fels and Gifsyprophage. The Fels prophage recombinase/invertases can be deleted usingthe pCVD442 homologous recombination system as described above for IS200elements. Deletion results in the inability to excise the phage DNA andtherefore is unable to undergo the lytic cycle or genetic recombination.

The Fels-1 invertase has a well known amino acid and DNA sequence. TheFels-2 recombinase/invertases also have known amino acid sequences, andDNA sequences.

2.13 Example of Genetic Isolation by Constitutive Expression of the P22Phage C2 Repressor.

See: Donnenberg and Kaper, 1991; Low et al. (Methods in MolecularMedicine, 2003), expressly incorporated herein by reference.

2.14 Example of Chromosomal Integration of a Synthetically Constructedavian Influenza Hemagglutinin Gene and Neuraminidase gene.

See: Donnenberg and Kaper, 1991; Low et al. (Methods in MolecularMedicine, 2003), expressly incorporated herein by reference.

2.15 Example of Determining Immune Response to H5N1 Expressing Bacteria.

Experimental determination of vaccine activity is known to those skilledin the art. By way of non-limiting example, determination of an antibodyresponse is demonstrated.

-   -   1) Vertebrate animals including mice, birds, dogs, cats, horses,        pigs or humans are selected for not having any known current or        recent (within 1 year) influenza infection or vaccination. Said        animals are pre-bled to determine background binding to, for        example, H5 and N1 antigens.    -   2) The Salmonella expressing H5 and N1 are cultured on LB agar        overnight at 37°. Bacteria expressing other H and or N antigens        may also be used.    -   3) The following day the bacteria are transferred to LB broth,        adjusted in concentration to OD₆₀₀=0.1 (˜2×10⁸ cfu/ml), and        subjected to further growth at 37° on a rotator to OD₆₀₀=2.0,        and placed on ice, where the concentration corresponds to        approx. 4×10⁹ cfu/ml.    -   4) Following growth, centrifuged and resuspended in 1/10 the        original volume in a pharmacologically suitable buffer such as        PBS and they are diluted to a concentration of 10⁴ to 10⁹ cfu/ml        in a pharmacologically suitable buffer on ice, warmed to room        temperature and administered orally or intranasally in a volume        appropriate for the size of the animal in question, for example        50 μl for a mouse or 10 to 100 ml for a human. The actual dose        measured in total cfu is determined by the safe dose as        described elsewhere in this application.    -   5) After 2 weeks, a blood sample is taken for comparison to the        pretreatment sample. A booster dose may be given. The booster        may be the same as the initial administration, a different        species, a different serotype, or a different flagellar antigen        (H1 or H2) or no flagellar antigen.    -   6) After an additional 2 to 4 weeks, an additional blood sample        may be taken for further comparison with the pretreatment and 2        week post treatment.    -   7) A comparison of preimmune and post immune antibody response        is preformed by immunoblot or ELISA. A positive response is        indicated by a relative numerical value 2× greater then        background/preimmune assay.        2.16 Example of Immunization with H5N1 Bacterial Vaccine        Strains.

An experiment to determine if H5N1 strains of Salmonella are capable ofproviding protection from challenge with the wildtype strain. Ducks areimmunized orally with 5×10⁹ cfu of bacteria when 4 weeks old, thenchallenged with the standard challenge model of avian influenza at 6weeks age.

Birds in Group A are immunized with empty vector. Group B receiveSalmonella H5N1. Group C is immunized with Salmnonella expressing theTamiflu resistant neuraminidase mutations. Birds in Group D are notimmunized. Each group is further divided into +/−Tamiflu treatment.Results of these experiments can be used to demonstrate theeffectiveness of the vaccine on Tamiflu resistant strain, with andwithout Tamiflu treatment.

Other Embodiments

Other embodiments are within the claims set forth below. For example,the host bacterium (the bacterium the chromosome of which is engineeredto encode a heterologous antigen) can be E. coli or any other entericbacterium, including Salmonella, Bordetella, Shigella, Yersenia,Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus, Providencia,Serratia, Plesiomonas, and Aeromonas, all of which are known or believedto similar to the promoters of E. coli and Salmonella. Also potentiallyuseful would be a bacille Calmette-Guerin (BCG) vaccine strainengineered to encode a heterologous antigen. The promoter used can benative to the species of the host bacterium, or can be a heterologouspromoter (i.e., from a species other than that of the host bacterium)engineered into the host bacterium along with the heterologous antigencoding sequence, using standard genetic engineering techniques. Multipleheterologous antigen coding sequences linked to the same or differentpromoter sequences can be inserted into a given chromosome, usingtechniques analogous to those set forth above, to produce a multivalentvaccine strain.

Those who practice in the field of prokaryotic gene expression willrealize that, while naturally-occurring promoter sequences arepreferred, synthetic sequences or a hybrid of two or more sequenceswould also be expected to be useful in the chromosomes of the invention.Alteration, addition or deletion of one or a few nucleotides within anaturally-occurring promoter sequence would generally not affect itsusefulness. The invention therefore encompasses promoters having suchinconsequential changes.

What is claimed is:
 1. A live Salmonella bacterium, configured to act asa vaccine, comprising: a mutation in a genetic locus of said bacteriumthat attenuates virulence of said bacterium in an animal host, such thatthe bacterium is capable of replicating but is safe to administer to theanimal host; wherein said live bacterium comprises Salmonella, whereinat least one IS200 element is deleted, and which constitutivelyexpresses P22 phage C2 repressor without dependence on a presence of anactivator or inducer molecule, to prevent new infections by bacteriaphage and prevent subsequent phage transductions by bacteria phage; aDNA construct comprising a promoter nucleotide sequence, a firstnucleotide sequence coding for a non-bacterial immunogenic polypeptide,wherein the nucleotide sequence coding for the non-bacterial immunogenicpolypeptide has at least one codon optimized for bacterial expressionwith respect to its non-bacterial counterpart, and a second nucleotidesequence coding for a bacterial secretion peptide sequence, wherein thefirst nucleotide sequence and second nucleotide sequence are in frameand code for a fusion peptide; said bacterial secretion peptide sequenceof the fusion peptide interacting with an available secretion mechanismof the bacterium, to cause a secretion of the fusion peptide from thebacterium in a form wherein the non-bacterial immunogenic polypeptideretains its immunogenicity after secretion substantially withoutinterference of the bacterial secretion peptide sequence; the bacteriumbeing configured to stably maintain the DNA construct and stably expressand secrete the fusion peptide from the bacterium over time; thebacterium being further configured to transiently infect a local tissueof the animal, secrete the fusion peptide, and raise an immune responseof the host animal to the non-bacterial immunogenic polypeptide,substantially without causing a persistent or systemic infection withthe bacterium or transducing other persistent bacteria to stably secretethe fusion peptide.
 2. The live bacterium according to claim 1, whereinsaid bacterium has sufficient deletions in bacteria phage or prophageelements and presence of at least one phage repressor which (a) enhancegenetic stability, (b) prevent phage excision, (c) prevent geneticrearrangement using bacteria phage or prophage elements, (d) reducecapacity for transduction of genes to other bacterial strains, and (e)prevents new infections by bacteria phage and further preventingsubsequent phage transductions by these phage.
 3. The live bacteriumaccording to claim 1, wherein the DNA construct is integrated into thechromosome of the bacterium.
 4. The live bacterium according to claim 1,wherein said bacterium is selected from the group consisting ofSalmonella enterica serovar Typhimurium (S. typhimurium), Salmonellamontevideo, Salmonella enterica serovar Typhi (S. typhi), Salmonellaenterica serovar Paratyphi B (S. paratyphi B), Salmonella entericaserovar Paratyphi C (S. paratyphi C), Salmonella enterica serovar Hadar(S. hadar), Salmonella enterica serovar Enteriditis (S. enteriditis),Salmonella enterica serovar Kentucky (S. kentucky), Salmonella entericaserovar Infantis (S. infantis), Salmonella enterica serovar Pullorum (S.pullorum), Salmonella enterica serovar Gallinarum (S. gallinarum),Salmonella enterica serovar Muenchen (S. muenchen), Salmonella entericaserovar Anaturn (S. anatum), Salmonella enterica serovar Dublin (S.dublin), Salmonella enterica serovar Derby (S. derby), Salmonellaenterica serovar Choleraesuis var. kunzendorf (S. cholerae kunzendorf),and Salmonella enterica serovar minnesota (S. Minnesota).
 5. The livebacterium according to claim 4, wherein said live bacterium is S.enterica serovar Typhimurium (S. typhimurium).
 6. The live bacteriumaccording to claim 1, wherein said mutation is in a genetic locusselected from the group consisting of phoP, phoQ, Mt, cya, crp, poxA,rpoS, htrA, nuoG, pmi, galE, pabA, pts, damA, purA, purB, purl, zwf,gua, cadA, rfic, rjb, rfa, ompR, msbB, Suwwan and combinations thereof.7. The live bacteria according to claim 1, wherein said bacterium has adeletion in the asd gene, and said non-bacterial immunogenic polypeptidecomprises a viral antigen or an immunogenic portion thereof.
 8. The livebacteria according to claim 7, wherein said bacterium secretes an fHlantigen or an immunogenic portion thereof, which is encoded on anantigen-expressing, multi-copy plasmid.
 9. The live bacterium accordingto claim 8, wherein an origin of replication of said multi-copy plasmidis a ColE1, pUC, M15, or pBR322 plasmid origin of replication.
 10. Thelive bacterium according to claim 1, wherein said bacterium isgenetically stabilized through deletion of sufficient IS200 elements andbacteria phage and prophage elements, to reduce their geneticrecombination and transduction potential with respect to a wild typeSalmonella of the same serovar.
 11. The live bacterium according toclaim 1, further comprising a pharmaceutical formulation adapted fororal administration of a plurality of the bacterium, to a human, toinduce a protective immune response to a non-bacterial organism which ischaracterized by an antigen corresponding to the non-bacterialimmunogenic polypeptide.
 12. The live bacterium according to claim 1,wherein the non-bacterial immunogenic polypeptide comprises an InfluenzaA hemagglutinen.
 13. The live bacterium according to claim 1, whereinthe non-bacterial immunogenic polypeptide comprises an Influenza Aneuraminidase.
 14. A live Salmonella bacterium, comprising: a deletionof at least one IS200 element; a P22 phage C2 repressor which isconstitutively expressed without dependence on a presence of anactivator or inducer molecule; a stabilized DNA construct having apromoter sequence and encoding a fusion peptide, said fusion peptidecomprising a bacterial secretion peptide portion and a non-bacterialimmunogenic polypeptide portion, wherein the DNA construct has anucleotide sequence coding for the non-bacterial immunogenic polypeptideportion which has at least one codon optimized for bacterial expressionwith respect to its non-bacterial counterpart, wherein the DNA constructis stabilized against transduction of other bacteria; and said bacteriumhaving a secretion mechanism which interacts with at least the bacterialsecretion peptide portion to cause a secretion of the fusion peptidefrom the bacterium; the bacterium having a genetic virulence attenuatingmutation and being adapted to act as an animal vaccine, to transientlyinfect a tissue of the animal, and cause an immune response to thenon-bacterial immunogenic polypeptide portion to cause an immunity inthe animal to a non-bacterial organism associated with the non-bacterialimmunogenic polypeptide portion.
 15. The live Salmonella bacteriumaccording to claim 14, wherein said bacterium has sufficient deletionsin bacteria phage or prophage elements and presence of at least onephage repressor which (a) enhance genetic stability, (b) prevent phageexcision, (c) prevent genetic rearrangement using bacteria phage orprophage elements, (d) reduce capacity for transduction of genes toother bacterial strains, and (e) prevents new infections by bacteriaphage and further preventing subsequent phage transductions by thesephage.
 16. The live Salmonella bacterium according to claim 14, whereinthe DNA construct is integrated into the chromosome of the bacterium.17. The live Salmonella bacterium according to claim 14, wherein saidbacterium is selected from the group consisting of Salmonella entericaserovar Typhimurium (S. typhimurium), Salmonella montevideo, Salmonellaenterica serovar Typhi (S. typhi), Salmonella enterica serovar ParatyphiB (S. paratyphi B), Salmonella enterica serovar Paratyphi C (S.paratyphi C), Salmonella enterica serovar Hadar (S. hadar), Salmonellaenterica serovar Enteriditis (S. enteriditis), Salmonella entericaserovar Kentucky (S. kentucky), Salmonella enterica serovar Infantis (S.infantis), Salmonella enterica serovar Pullorum (S. pullorum),Salmonella enterica serovar Gallinarum (S. gallinarum), Salmonellaenterica serovar Muenchen (S. muenchen), Salmonella enterica serovarAnaturn (S. anatum), Salmonella enterica serovar Dublin (S. dublin),Salmonella enterica serovar Derby (S. derby), Salmonella entericaserovar Choleraesuis var. kunzendorf (S. cholerae kunzendorf), andSalmonella enterica serovar minnesota (S. Minnesota), and the geneticvirulence attenuating mutation comprises a mutation in at least onegenetic locus selected from the group consisting of phoP, phoQ, Mt, cya,crp, poxA, rpoS, htrA, nuoG, pmi, galE, pabA, pts, damA, purA, purB,purl, zwf, gua, cadA, rfic, rjb, rfa, ompR, msbB, and Suwwan.
 18. Thelive Salmonella bacteria according to claim 14 wherein saidnon-bacterial immunogenic polypeptide portion comprises an AvianInfluenza antigen or an immunogenic portion thereof.
 19. The liveSalmonella bacterium according to claim 14, further comprising apharmaceutical formulation adapted for oral administration of aplurality of the bacterium, to a human, to induce a protective immuneresponse to a non-bacterial organism which is characterized by anantigen corresponding to the non-bacterial immunogenic polypeptideportion.
 20. A live bacterium, comprising: a stabilized DNA constructintegrated into a Salmonella bacteria chromosome, having a promotersequence and encoding a fusion peptide, said fusion peptide comprising abacterial secretion peptide portion and an Influenza immunogenicpolypeptide portion, wherein the DNA construct has a nucleotide sequencecoding for the Influenza immunogenic polypeptide portion which has atleast one codon optimized for Salmonella expression with respect to itsInfluenza counterpart, wherein the DNA construct is stabilized againsttransduction of other bacteria; genetic modifications with respect to awild type Salmonella effective to substantially prevent new infectionsby bacteria phage and prevent subsequent phage transductions by bacteriaphage, comprising at least a deletion of at least one IS200 element ofthe live Salmonella bacteria chromosome, and a constitutively expressedP22 phage C2 repressor without dependence on a presence of an activatoror inducer molecule; and said bacterium having a secretion mechanismwhich interacts with at least the bacterial secretion peptide portion tocause a secretion of the fusion peptide from the bacterium; thebacterium having a genetic virulence attenuating mutation and being ananimal Influenza vaccine, which causes a localized transient intestinalepithelial tissue infection of the animal, and induces an immuneresponse to the Influenza immunogenic polypeptide portion to create animmunity in the animal to an Influenza strain corresponding to theInfluenza immunogenic polypeptide portion.